Process for the enzymatic synthesis of beta-nicotinamide mononucleotide and its polyenzymatic combination

Abstract

This invention provides a method for synthesizing β-nicotinamide mononucleotide (NMN) and its multi-enzyme-based synthesis, belonging to the field of bioengineering technology. The multi-enzyme-based synthesis method employs a three-enzyme cascade system composed of nicotinamide phosphoribosyltransferase (NAMPT), ribokinase (Rbks), and phosphoribosyl pyrophosphate synthase (PRPS). PRPS synthesizes PRPP in situ using ATP and R5P as substrates. NAMPT and Rbks then synthesize the target product β-nicotinamide mononucleotide using the in-situ synthesized PRPP and nicotinamide as substrates, thus eliminating the need for the substrate PRPP and effectively reducing production costs. The NAMPT enzyme exhibits good activity and thermal stability at room temperature, effectively improving the yield and purity of β-nicotinamide mononucleotide, achieving a conversion rate of over 80%, resulting in a final purified β-nicotinamide mononucleotide with high biological activity and stability.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to a β-nicotinamide mononucleotide and its multi-enzyme-level synthesis method. Background Technology

[0002] β-Nicotinamide mononucleotide (β-NMN) has the molecular formula C 11 H 15 N2O8P, with a molecular weight of 334.2192 g / mol, is composed of phosphate groups, ribose, and nicotinamide groups. Research by David Sinclair's team at Harvard University and other scholars has shown that β-NMN supplementation can rapidly increase the body's NAD+ levels, which gradually decline with age, thereby effectively activating the Sirtuins ("longevity proteins") family and slowing down the aging process. Multiple animal model studies have further confirmed that β-NMN intake can significantly improve metabolic function and extend healthy lifespan.

[0003] The NAD+ salvage pathway includes the nicotinamide (NAM) rescue pathway: NAM reacts with phosphoribosyl pyrophosphate under the catalysis of nicotinamide phosphoribosyltransferase (NAMPT) to undergo a phosphoribosyl transfer reaction, completely generating high-purity and highly active β-NMN and pyrophosphate (PPi). This NAM rescue pathway is named the NAMPT route after the key enzymes involved. Because it directly utilizes NAM as a substrate, the NAMPT route is considered a "golden route" that better aligns with the core endogenous metabolic logic of the human body. However, the substrate 5-phospribose-1-pyrophosphate (PRPP) required for the synthesis of β-NMN via the NAMPT route is expensive and unstable, resulting in excessively high costs for large-scale production. This makes the NAMPT route costly to synthesize high-purity and highly active β-NMN, hindering large-scale production.

[0004] To address this, Chinese invention patent application CN 118240854 A discloses a method for synthesizing a four-gene co-expression vector and engineered bacteria using a whole-enzyme method. The specific steps are as follows: constructing a dual-gene expression vector, constructing a four-gene co-expression vector pRSFT7, constructing a four-gene co-expression mutant vector, inducing the construction of the engineered bacteria expressing the four-gene co-expression, and developing the enzyme. This invention achieves four-gene co-expression using a single vector, requiring only one engineered bacteria to ferment and express the necessary enzymes. This simplifies the whole-enzyme method for synthesizing NMN, reduces enzyme production costs, avoids the use of expensive raw material PRPP, and regenerates ATP (adenine triphosphate) during the reaction process, significantly reducing raw material costs. This invention patent application discloses a 3L reaction system with a maximum NMN yield of 17.64 g / L and a NAM molar conversion rate of 34.19%. Therefore, while the enzyme production cost is reduced in this invention patent application, the NMN yield needs further improvement. Summary of the Invention

[0005] In view of this, the main objective of this invention is to provide a method for synthesizing β-nicotinamide mononucleotide and its multi-enzyme cascade. This synthesis method employs a three-enzyme cascade system consisting of nicotinamide phosphoribosyltransferase (NAMPT), ribokinase (Rbks), and phosphoribosyl pyrophosphate synthase (PRPS). PRPS synthesizes PRPP in situ using ATP and R5P as substrates. NAMPT and Rbks use the in-situ synthesized PRPP and nicotinamide as substrates to synthesize the target product, β-nicotinamide mononucleotide. This method eliminates the need for the substrate PRPP, effectively reducing production costs, and also achieves efficient production of β-NMN.

[0006] Specifically, in a first aspect, the present invention provides a multi-enzyme-based synthesis method for β-nicotinamide mononucleotide, comprising: adding PRPS enzyme solution, NAMPT enzyme solution, Rbks enzyme solution, and hPrs enzyme solution to a substrate solution to form a β-NMN reaction system, wherein the β-NMN reaction system is fermented at 35-40 °C, pH 7.6-8.5, and 250-350 r / min for 200-550 min; wherein the substrate solution comprises nicotinamide, ribose 5-phosphate (R5P), and ATP, and, based on the final concentration, the β-NMN reaction system comprises 0.5-3 mM nicotinamide, 0.5-3 mM ribose 5-phosphate, 10-30 mM ATP, 10-15 mM MgCl2, and 45-55 mM ATP. In the Tris-HCl buffer, the concentration ratio of nicotinamide phosphoribosyltransferase (NAMPT), ribokinase Rbks, and phosphoribosyl pyrophosphate synthase (PRPS) in the β-NMN reaction system is 0.8-1.2 : 0.8-1.2 : 0.8-1.2. The nucleotide sequence of nicotinamide phosphoribosyltransferase (NAMPT) is shown in SEQ ID NO. 1, and the ribokinase Rbks is derived from... Escherichia coli Rbks (eRbks) or source H. sapiens The amino acid sequence of the phosphoribosyl pyrophosphate synthase PRPS is shown in SEQ ID NO. 2.

[0007] In one specific embodiment, the method for preparing the NAMPT enzyme solution includes: culturing the engineered strain BL21(DE3)-NAMPT expressing the nicotinamide phosphoribosyltransferase NAMPT in a culture medium at 36-38℃ and 130-150 rpm for 7-9 hours; and then measuring the OD... 600When the concentration reaches the range of 0.6-0.8, cool to 15-17 ℃ and add IPTG at a concentration of 0.15-0.25 mM. Then, perform percolation expression for 14-18 h to obtain NAMPT fermentation broth. Centrifuge the NAMPT fermentation broth to collect the precipitate and obtain BL21(DE3)-NAMPT wet cells. First, add Tris-NaCl diluent to the BL21(DE3)-NAMPT wet cells and re-vortex. Then, add 0.9-1.1 mM PMSF for sonication to disrupt the cells. Centrifuge again at 11000-13000 rpm for 5-15 min and collect the supernatant to obtain a crude protein solution containing NAMPT. Elute the crude protein solution containing NAMPT with imidazole solution at 0℃-4℃ and dialyze to remove imidazole to obtain the NAMPT enzyme solution. The Tris-NaCl diluent contains 24-26 mM Tris and 145-155 mM NaCl. Preferably, the NAMPT enzyme solution has an enzyme activity of 30-35 U / mL.

[0008] The preparation method of the PRPS enzyme solution includes: constructing recombinant Escherichia coli BL21(DE3)-PRPS expressing the phosphoribosyl pyrophosphate synthase PRPS; placing the recombinant Escherichia coli BL21(DE3)-PRPS in resistant LB liquid medium and fermenting it under the conditions of 0.15-0.25 mmol / L total IPTG concentration, 35-40 ℃, and 150-250 r / min to obtain PRPS fermentation broth; centrifuging the PRPS fermentation broth to collect the precipitate and obtain BL21(DE3)-PRPS wet cells; washing the BL21(DE3)-PRPS wet cells with distilled water and adding buffer. BL21(DE3)-PRPS cells were suspended in buffer A; the recombinant E. coli BL21(DE3)-PRPS cells were disrupted using an ultrasonic cell disruptor under ice bath conditions to obtain a PRPS cell suspension; the PRPS cell suspension was subjected to freeze centrifugation, and the precipitate was discarded to obtain a crude protein solution containing PRPS; the crude protein solution of PRPS was purified by Ni affinity chromatography, wherein imidazole solution was used for elution, and the imidazole was removed by dialysis to obtain the PRPS enzyme solution; wherein the buffer A contained 19-21 mM imidazole, 24-26 mM Tris, and 140-160 mM NaCl.

[0009] The preparation method of the Rbks enzyme solution includes: constructing recombinant Escherichia coli BL21(DE3)-Rbks expressing the ribokinase Rbks; fermenting and culturing the recombinant Escherichia coli BL21(DE3)-Rbks, collecting the precipitate by centrifugation to obtain BL21(DE3)-Rbks wet cells; resuspending the BL21(DE3)-Rbks wet cells, sonicating and centrifuging to obtain the supernatant to obtain a crude protein solution containing Rbks; purifying the crude protein solution containing Rbks by column chromatography, wherein imidazole solution is used for elution, and imidazole is removed by dialysis to obtain the Rbks enzyme solution.

[0010] To further improve the yield of β-NMN, the fermentation time of the β-NMN reaction system is 200-300 min.

[0011] Furthermore, to obtain β-NMN with a purity of over 99%, the multi-enzyme-level synthesis method further includes: obtaining β-NMN fermentation broth after the β-NMN reaction system has completed the reaction; separating, purifying, and drying the β-NMN fermentation broth to obtain β-NMN.

[0012] To further obtain β-NMN with a purity of not less than 99.9%, the β-NMN fermentation broth was separated and purified using HPLC-UV (high performance liquid chromatography coupled with ultraviolet detection). The HPLC detection conditions included: an Agilent ZORBAX SB-C18 column; mobile phase A consisting of phosphate at pH 5.5-6.0; mobile phase B consisting of a mixture of methanol and formic acid, with formic acid accounting for 0.008-0.012% by mass; isocratic elution; and elution time of 5-20 min.

[0013] Furthermore, to ensure that the β-NMN pure product is fully dried, the drying method of the β-NMN pure product includes: drying the β-NMN purified by HPLC-UV method in a vacuum drying oven at 40-60 ℃ for 8-24 h to obtain the β-NMN pure product.

[0014] Secondly, the present invention also provides a pure β-nicotinamide mononucleotide obtained by the above-described synthesis method, with a purity ≥99.9% and a moisture content not exceeding 1%. Furthermore, the purity of the β-NMN pure product reaches 100%. Compared with the prior art, the above-described technical solution provided by the present invention has the following advantages: Low cost: This invention uses a three-enzyme cascade of PRPS, NAMPT, and Rbks, combined with a "one-pot" method to prepare β-NMN based on the NAMPT route. This method uses on-site self-made PRPS enzyme solution to achieve in-situ generation of PRPP. Compared with directly purchasing PRPP products, it achieves a significant reduction in the production cost of β-NMN, with a cost reduction of more than 96%, while also achieving efficient synthesis of β-NMN. High yield and superior performance: This invention uses a special NAMPT enzyme solution with good activity and good thermal stability at room temperature, which can effectively improve the yield and purity of β-NMN, with a conversion rate of over 80%; in addition, based on the on-site self-made high-activity PRPS enzyme solution, NAMPT enzyme solution, and Rbks enzyme solution, the final β-NMN pure product has high biological activity and stability. High purity: This invention uses HPLC-UV method to purify the synthesized β-NMN fermentation broth and adds formic acid to the mobile phase, so that the product purity can be ≥99.9% after only one purification, reaching the pharmaceutical grade standard, and the yield is 80-90%. Attached Figure Description

[0015] Figure 1 SDS-PAGE images of the purified PRPS enzyme solution provided for the examples (lane 1: cell; 2: cell disruption; 3: first wash; 4: second wash; 5: third wash; 6: first elution; 7: second elution). Figure 2 The image shows the results of the fluorescence colorimetric reaction of the PRPS protein provided in Example 1. Figure 3 The figure shows the effect of PRPS enzyme on the synthesis of β-NMN. Figure 4 Chromatograms of β-NMN fermentation broth before and after purification; Figure 5 The graph shows the stability results of β-NMN prepared in Example 3. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0017] In this invention, there are no special requirements for the type of expression plasmid. The construction method for expressing the target gene in *E. coli* can employ various methods commonly used in the art, such as ligating the target gene into a vector after enzyme digestion, replacing the promoter, gene knockout, gene mutation, etc., which will not be elaborated further. Unless otherwise specified in the examples, conventional conditions should be followed, such as those described in *Molecular Cloning: A Laboratory Manual (3rd Edition)*, edited by J. Sambrook et al., Science Press, 2002; *Cellular Laboratory Manual*, edited by DL Spector et al., Science Press, 2001; or the manufacturer's recommended conditions. Unless otherwise specified, "M" in this invention refers to the concentration unit "mol / L," such as "mM" which refers to the concentration unit "mmol / L."

[0018] Unless otherwise specified, all reagents or instruments used in the following embodiments, unless otherwise indicated by the manufacturer, are commercially available products. The ribokinase Rbks used in the following examples is Rbks human NP071411.1, and the amino acid sequence of phosphoribosyl pyrophosphate synthase PRPS is shown in SEQ ID NO. 2.

[0019] The plasmids, restriction enzymes, PCR enzymes, column-based DNA extraction kits, and DNA gel recovery kits used in the following examples are commercial products, and the specific operations were performed according to the kit instructions. Specifically, the plasmid pET-30a used in the following examples is P. calidifontis ABO08552.1.

[0020] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in this invention all employ conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. Specifically, they can be performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0021] In addition, the "water" mentioned in this invention includes any feasible water that can be used in the art, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, and purified water.

[0022] The following examples use Reagent formulation as follows: Buffer A (for resuspending crude PRPS enzyme solution): 20 mM imidazole, 25 mM Tris, 150 mM NaCl; Buffer B (for rinsing PRPS pure enzyme solution): 150 mM imidazole, 25 mM Tris, 150 mM NaCl.

[0023] In the following embodiments, During the HPLC-UV method for detecting β-NMN β-Nicotinamide mononucleotide (β-NMN, ≥95%) produced by Sigma-Aldrich was selected as the reference standard. The β-NMN standard was dissolved in 50% methanol aqueous solution to obtain a solution with a concentration of 5 mg / mL. The solution was then diluted to 100 μg / mL with 50% methanol aqueous solution to obtain the standard dilution. After filtration through a 0.22 μm organic phase filter membrane, the solution was transferred to an autosampler vial for HPLC-UV analysis.

[0024] The following examples are used Preparation method of NAMPT enzyme solutionThe process includes: codon optimization of the NAMPT enzyme encoding gene with the amino acid sequence shown in SEQ ID NO. 1, resulting in the nucleotide sequence shown in SEQ ID NO. 3. This nucleotide sequence was digested with restriction endonucleases NdeI and NotI and ligated into the vector pET-30a, which had also been digested with the same restriction endonucleases NdeI and NotI, to obtain the plasmid pET30a-nampt. The constructed recombinant plasmid pET30a-nampt was transformed into BL21(DE3) competent cells and cultured overnight at 37°C inverted on LB plates containing Kan resistance. Positive monoclonal cells were selected to obtain the engineered bacterium BL21(DE3)-NAMPT. The engineered bacterium BL21(DE3)-NAMPT was inoculated into 10 mL centrifuge tubes containing Kan resistance LB medium and cultured in a shaker at 37°C and 180 rpm for 8 h to obtain the seed culture. The seed culture was transferred at a 2% inoculation ratio to 1 L LB liquid medium containing resistant Kan, and cultured in a shaker at 37°C and 140 rpm for 8 h. When the OD600 reached 0.6-0.8, the temperature was lowered to 16°C, IPTG was added at a concentration of 0.2 mM, and expression was induced for 16 h to obtain NAMPT fermentation broth. The NAMPT fermentation broth was centrifuged at 4°C and the cells were collected to obtain BL21(DE3)-NAMPT wet cells. The BL21(DE3)-NAMPT wet cells were re-vortexed with 25 mM Tris and 150 mM NaCl (pH=7.5), and 1 mM PMSF was added before sonication to disrupt the cells. After centrifugation at 12000 rpm for 10 min, the supernatant was obtained as the crude protein solution containing NAMPT. The crude protein solution containing NAMPT was co-incubated with a certain amount of 6x His-tag Purification Resin in a rotary shaker at 5°C. The eluent was washed sequentially with a low-concentration imidazole solution (21 mM imidazole, 26 mM Tris, 155 mM NaCl) and a high-concentration imidazole solution (145 mM imidazole, 24 mM Tris, 145 mM NaCl). The obtained protein solution was dialyzed at 5°C using a 3500 kD dialysis bag in 26 mM Tris and 155 mM NaCl (pH 7.6) to remove imidazole, yielding pure NAMPT enzyme solution.

[0025] Preparation method of Rbks enzyme solutionThe procedure included: codon optimization of the coding sequence of the Rbks human NP 071411.1 gene. NdeⅠ and NotⅠ restriction enzyme sites were introduced upstream and downstream of the gene sequence, respectively, and a 6×His tag was added to the N-terminus. The modified synthetic gene was cloned into the pET-30a(+) vector between the NdeⅠ and NotⅠ sites, and the resulting recombinant plasmid was named pET-30a(+)-hRbks. Double enzyme digestion confirmed the correct construction of the recombinant plasmid. Agarose gel electrophoresis showed consistency with the target gene hRbks, indicating successful construction of pET-30a(+)-hRbks. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37°C on kanamycin-resistant LB agar plates. Single clones were screened, and after expansion culture, the plasmid was extracted and double enzyme digestion was performed for verification. Finally, the correctly identified strain BL21(DE3)-Rbks was stored in glycerol tubes at -80°C. Activated recombinant *E. coli* BL21(DE3)-Rbks were picked and single colonies were inoculated into 10 mL of LB broth containing 50 μg / L kanamycin and cultured at 37 °C and 200 r / min for 12–15 h. The overnight culture was then transferred at a 2% inoculum to 100 mL of LB broth containing 50 μg / L kanamycin and cultured at 37 °C and 200 r / min until the OD600 reached approximately 0.6–0.8. 1 mol / L IPTG was added to a final concentration of 0.20 mmol / L, and the culture was induced at 16 °C and 180 r / min for 21 h to obtain the *Rbks* fermentation broth. The *Rbks* fermentation broth was centrifuged at 7800 r / min for 7 min, and the supernatant was discarded to obtain wet *BL21(DE3)-Rbks* cells. The wet *BL21(DE3)-Rbks* cells were washed once with distilled water and then resuspended in 10 mL of Buffer A. Recombinant *E. coli* was disrupted using an ultrasonic cell disruptor under ice bath conditions: 6 mm amplitude transducer, 40 min, 10 mL bacterial suspension. The disrupted suspension was then centrifuged at 10000 r / min, 4 ℃, for 30 min to separate the supernatant and precipitate. The precipitate was discarded, yielding a crude protein solution containing Rbks. This crude protein solution containing Rbks was purified by column chromatography. A Ni column (His Tag™ FF) was installed, and the column was washed with 4 column volumes of ultrapure water until baseline level was reached. The column was then washed with Buffer B until baseline level was reached, followed by washing with Buffer A until baseline level was reached. The sample was then loaded at a constant flow rate of 3 mL / min, and finally eluted and collected with 80% Buffer B.Wash with 5 column volumes of ultrapure water until the baseline is level, and finally wash with 20% ethanol to preserve the column and system. Remove the column, shut off the system, and store the removed column in a 4 ℃ refrigerator to obtain the Rbks pure enzyme solution. Example 1: Homemade PRPS enzyme solution

[0026] This embodiment provides a method for preparing PRPS enzyme solution, including the following steps: Construction of recombinant *E. coli* BL21(DE3)-PRPS: Codon optimization was performed on the encoding gene of the PRPS enzyme, as shown in SEQ ID NO. 2, to obtain the nucleotide sequence shown in SEQ ID NO. 4. NdeⅠ and NotⅠ restriction enzyme sites were introduced upstream and downstream of this nucleotide sequence, respectively, and a 6×His tag was added to the N-terminus. The modified synthetic gene was cloned into the pET-30a(+) vector between the NdeⅠ and NotⅠ sites, and the resulting recombinant plasmid was named pET-30a(+)-PRPS. Double enzyme digestion confirmed the correct construction of the recombinant plasmid. Agarose gel electrophoresis showed consistency with the target gene PRPS, indicating successful construction of pET-30a(+)-PRPS. The verified recombinant plasmid was transformed into E. coli BL21(DE3) competent cells and cultured overnight at 37°C upside down on kanamycin-resistant LB agar plates. Single clones were screened, and after expansion culture, the plasmid was extracted and verified by double enzyme digestion. Finally, the correctly identified strain BL21(DE3)-PRPS was stored in glycerol tubes at -80°C.

[0027] Obtaining wet bacterial cells: Activated recombinant *E. coli* BL21(DE3)-PRPS was picked and a single colony was inoculated into 10 mL of LB broth containing 50 μg / L kanamycin and cultured at 37 °C and 200 r / min for 12–15 h. The overnight culture was then transferred at a 2% inoculum to 100 mL of LB broth containing 50 μg / L kanamycin and cultured at 37 °C and 200 r / min until the OD600 reached approximately 0.6–0.8. 1 mol / L IPTG was added to a final concentration of 0.20 mmol / L, and the culture was induced at 16 °C and 180 r / min for 21 h to obtain the PRPS fermentation broth. The PRPS fermentation broth was centrifuged at 7800 r / min for 7 min, and the supernatant was discarded to obtain the BL21(DE3)-PRPS wet bacterial cells.

[0028] PRPS crude enzyme solution: BL21(DE3)-PRPS wet cells were washed once with distilled water and then resuspended in 10 mL of Buffer A. Recombinant E. coli were disrupted using an ultrasonic cell disruptor under ice bath conditions: 6 mm amplitude rod, 40 min, 10 mL cell suspension. The disrupted suspension was then centrifuged at 10000 r / min, 4 ℃, for 30 min to separate the supernatant and precipitate. The precipitate was discarded to obtain the PRPS crude enzyme solution.

[0029] PRPS purified enzyme solution: The crude PRPS enzyme solution was purified using Ni affinity chromatography. A Ni column (His Tag™ FF) was assembled, and the column was washed with 4 column volumes of ultrapure water until baseline level was reached. This was followed by washing with Buffer B buffer until baseline level was reached, then with Buffer A buffer until baseline level was reached. The sample was then loaded at a constant flow rate of 3 mL / min. Finally, the target protein was eluted and collected with 80% Buffer B buffer. The column was washed with 5 column volumes of ultrapure water until baseline level was reached, and finally washed with 20% ethanol. The column and system were then stored. The column was removed, the system was shut off, and the removed column was stored at 4 °C to obtain the purified PRPS enzyme solution.

[0030] The purified PRPS enzyme solution was analyzed by SDS-PAGE, as follows: Figure 1 As shown. Figure 1 The SDS-PAGE image shown indicates that the PRPS enzyme appears as a single band after SDS-PAGE analysis, and the extracted PRPS molecular weight is approximately 61 kDa.

[0031] The effect of PRPS enzyme on the synthesis of β-NMN To further verify the effect of PRPS on β-NMN synthesis, this experiment designed multiple different enzyme reaction systems and used fluorescence method to determine the β-NMN yield to reflect the PRPS enzyme activity and function. The specific experimental details are as follows: Standard curve: Prepare several 69 μL β-NMN aqueous solutions with concentration gradients of 0-1 μmol / L. Add 27.7 μL of 2 mol / L KOH to each centrifuge tube, followed by 27.7 μL of 20% acetophenone. Vortex briefly to mix, then place in an ice bath for 2 min. Add 125 μL of 88% formic acid, centrifuge briefly at low speed, and react in a constant temperature shaker at 37℃ for 10 min. Transfer 240 μL of the liquid from each centrifuge tube to the wells of a black 96-well plate. Measure fluorescence using a Tecan multi-plate reader at excitation light of 382 nm and emission light of 445 nm. Use pure water as a blank control. Plot a standard curve based on β-NMN concentration and corresponding fluorescence values, as shown below. Figure 2 As shown.

[0032] Enzyme Reaction System 1: PRPS Enzyme Reaction System: Add the following to a 1.5 mL centrifuge tube: 3.45 μL 1 mol / L Tris-HCl, 1.38 μL 1% BSA, 0.83 μL 1 mol / L MgCl2, 1.38 μL 0.2 mol / L ATP, 0.28 μL 0.1 mol / L ribose-5-phosphate, 1.38 μL 0.1 mol / L dithiothreitol, 58.10 μL purified water, 1.5 μL of nampt enzyme solution diluted by a certain factor, 1.5 μL of hRbks enzyme solution diluted by a certain factor, 1.5 μL of PRPS enzyme solution diluted by a certain factor, and 0.69 μL 200 μmol / L NAM, with the concentration ratio of nampt, hRbks, and PRPS being 1:1:1. React in a constant temperature shaker at 45℃ for 15 min. A reaction system using pure water instead of enzyme solution was set up as a blank control.

[0033] Enzyme reaction system 2 is basically the same as the PRPS enzyme reaction system described above, except that 1.5 μL of a diluted hPrs pure enzyme solution is added to this system, and the concentration ratio of nampt, hRbks, PRPS, and hPrs in this system is 1:1:1:2; other parameters remain unchanged. The enzyme hPrs is another phosphoribosyl pyrophosphate synthase: *e. coli* POA9J6Prs human P60891, and the preparation method of the hPrs pure enzyme solution is the same as that of the PRPS pure enzyme solution provided in this embodiment.

[0034] Enzyme reaction system 3 is basically the same as enzyme reaction system 2 above, except that PRPS enzyme is omitted in this enzyme reaction system; the concentration ratio of nampt, hRbks and hPrs in this reaction system is 1:1:2; everything else remains the same.

[0035] Enzyme reaction system 4 is basically the same as the PRPS enzyme reaction system described above, except that: the hRbks enzyme and PRPS enzyme are omitted in this enzyme reaction system, and only nampt enzyme is added; 1.38 μL of 0.1 mol / L PRPP substrate is used to replace the substrates ATP and 5-phosphate ribose; everything else remains the same.

[0036] To stop the enzyme reaction: inactivate the enzymes by placing the centrifuge tube in a 95°C water bath for 1 minute.

[0037] The reaction of the β-NMN derivative product: Add 27.7 μL of 2 mol / L KOH to a centrifuge tube, then add 27.7 μL of 20% acetophenone, briefly vortex to mix, and place in an ice bath for 2 min. Add 125 μL of 88% formic acid, briefly centrifuge at low speed, and react in a constant temperature shaker at 37 ℃ for 10 min.

[0038] Fluorescence measurement: Pipette 240 μL of liquid from a centrifuge tube into the wells of a black 96-well plate. Measure the fluorescence intensity using a Tecan multi-mode microplate reader at excitation light of 382 nm and emission light of 445 nm. The results are as follows: Figure 3 As shown.

[0039] Figure 3 This indicates that the purified PRPS proteins provided in this embodiment are all active. Both enzyme reaction systems 1 and 2 showed positive fluorescence for β-NMN after the addition of the self-made PRPS enzyme. The β-NMN yield in enzyme reaction system 2 was slightly higher than that in enzyme reaction system 1, but this difference was negligible, indicating that the human phosphoribosyl pyrophosphate synthase (hPrs) has little effect on β-NMN synthesis. Enzyme reaction system 3, which added hPrs but not PRPS enzyme, had a relatively low β-NMN yield, less than 1 / 8 of that produced in enzyme reaction system 1. This demonstrates that the activity of the self-made PRPS enzyme in this embodiment is far superior to that of hPrs enzyme. Enzyme reaction system 1 is essentially the same as the synthesis of β-NMN directly using commercial PRPP substrate (enzyme reaction system 4), thus proving that PRPS protein has a significant impact on β-NMN synthesis, enabling in-situ laboratory production of PRPP and its successful application in the β-NMN synthesis process. Example 2: Synthesis method of β-NMN

[0040] This embodiment provides a multi-enzyme-based synthesis method for β-nicotinamide mononucleotide, comprising the following steps: adding the crude PRPS enzyme solution, NAMPT enzyme solution, and Rbks human NP 071411.1 obtained in Example 1 to a substrate solution to form a β-NMN reaction system. The β-NMN reaction system is fermented at 37 °C, pH 8.0, and 300 r / min for 250 min to obtain a β-NMN fermentation broth. The concentration ratio of enzymes NAMPT, Rbks, and PRPS in the β-NMN reaction system is 1:1:1 based on the final concentration, and includes 1 mM nicotinamide, 20 mM ATP, 1 mM R5P, 12.5 mM MgCl2, and 50 mM Tris-HCl buffer. Example 3 Purification of β-NMN fermentation broth

[0041] This embodiment provides a method for preparing β-NMN, comprising: firstly, separating and purifying the β-NMN fermentation broth obtained in Example 2 using HPLC-UV method, and then drying it in a vacuum drying oven at 50 °C for 16 h to obtain pure β-NMN. The HPLC-UV detection method includes: diluting the β-NMN fermentation broth obtained in Example 2 five times with 50% methanol aqueous solution, filtering it through a 0.22 μm organic phase filter membrane, and transferring it to an autosampler vial for HPLC-UV analysis. HPLC-UV analysis was performed on an Agilent 1200 Series HPLC system (Agilent Technologies, Santa Clara, CA, USA), which is equipped with a binary pump, autosampler, column oven, and UV detector. Chromatographic separation was performed using an Agilent ZORBAX SB-C18 column (250 mm × 4.6 mm, 5 μm). Mobile phase A was 10 mM phosphate buffer at pH 5.86; mobile phase B was a mixture of methanol and formic acid, with formic acid comprising 0.01% by mass. Isocratic elution was used, with mobile phase B comprising 85%, elution time 15 min, and flow rate 0.8 mL / min. The injection volume was 10 μL, and the column temperature was maintained at 30℃. The detection wavelength was set to 260 nm, and data acquisition and analysis were performed using Agilent ChemStation software (version B.04.03, Agilent Technologies, USA). The chromatographic separation parameters of the β-NMN reference standard are shown in Table 1. The HPLC chromatograms of the β-NMN fermentation broth before and after purification in this embodiment are shown in Table 1. Figure 4 As shown.

[0042] Table 1 Chromatographic separation parameters of β-NMN reference standard Standard products Retention time / min Retention time RSD / % Peak area / mAU Peak area RSD / % Theoretical number of plates / N Peak matching degree β-NMN 2.87 0.21 86.2556 0.52 17786 996 To separate the β-NMN sample from other components in the reaction system as much as possible, the mobile phase solution ratio was first optimized. The β-NMN sample was dissolved in a 50% methanol-water solution to prepare the test solution. β-NMN was then separated chromatographically using isocratic separation methods with 95% mobile phase B, 85% mobile phase B, and 50% mobile phase B (V / V). Peak assignment was confirmed using a single standard method, thus determining the optimal mobile phase ratio for detection.

[0043] As the methanol content in the mobile phase increases, the elution time of β-NMN gradually shifts later. When β-NMN is eluted at a 50% mobile phase ratio, it cannot be effectively separated from the matrix. While elution at a 95% mobile phase ratio B results in a later elution time for β-NMN, affecting the efficiency of the analytical method and also accompanied by peak broadening. The best separation effect is achieved when eluting at a 85% mobile phase ratio B, with a shorter elution time for β-NMN and a peak shape that largely conforms to a Gaussian distribution. This effectively separates impurity peaks in the matrix from the analyte peak, reducing the possibility of interference from impurity peaks and eliminating the need for purification of the extract. The overall analytical time can be controlled within 20 minutes, making the operation simple, time-saving, and improving analytical detection efficiency. For reaction samples with a basically complete reaction, it can be seen that elution at a 85% mobile phase ratio B provides the best separation effect, peak shape, and analytical efficiency. Therefore, elution at a 85% mobile phase ratio B is used for subsequent experiments.

[0044] Compared to mobile phase B without formic acid, the addition of 0.01% formic acid in this embodiment significantly improved the peak shapes of each analyte and kept the entire analysis time within 15 minutes, greatly enhancing analytical efficiency. This is likely because the addition of formic acid inhibited the dissociation of β-NMN, reducing its retention on the HPLC packing material.

[0045] like Figure 4 As shown, contaminating proteins in the β-NMN fermentation broth were washed away during purification, reducing their degradation of the β-NMN product. Furthermore, the β-NMN fermentation broth exhibited a high peak area at 4 min, suggesting that this peak represents other byproducts generated from the conversion of other contaminating proteins in the crude PRPS and NAMPT enzyme solutions using the substrate ATP. In contrast, the peak area of ​​the purified β-NMN sample at this point was significantly reduced, further demonstrating the relative purification of the β-NMN sample. Therefore, using the HPLC-UV method of this embodiment for a single separation and purification of the β-NMN fermentation broth resulted in a β-NMN sample yield of 54.17 g / L, a purity of 100%, and a yield of 87.2%.

[0046] Cost estimates for synthesizing PRPP based on self-made PRPS: PRPP synthesis requires D-ribose, ATP, phosphate, etc. The market price of D-ribose is approximately 500-800 RMB / kg, ATP approximately 2000-3000 RMB / kg, and phosphate is relatively inexpensive, at approximately 50-100 RMB / kg. Based on the calculation that producing 1 kg of PRPP requires 1.4-1.6 kg of D-ribose, 1.8-2.2 kg of ATP, and a suitable amount of phosphate, the cost is approximately 2800-3200 RMB / kg (0.0028-0.0032 RMB / mg), representing a cost reduction of over 99.9%. The estimated cost of synthesizing β-NMN based on commercially available PRPP is 35000-45000 RMB / kg. The estimated cost of synthesizing β-NMN using the NAMPT multi-enzyme cascade catalytic system provided in this example is 800-1300 RMB / kg, a cost reduction of over 96% compared to the cost based on commercially available PRPP. Example 4: Quality Testing and Evaluation of β-NMN

[0047] This embodiment tests and evaluates the pure β-NMN obtained in Example 3, specifically including the following aspects: 4.1 Appearance Characterization and Solubility Evaluation Standards Sensory indicators, as the primary and intuitive criteria for evaluating the quality of NMN products, not only reflect the initial effectiveness of the synthesis process but also preliminarily determine the purification depth and physicochemical state of the product. This project established appearance characterization and solubility evaluation standards in accordance with the Chinese Pharmacopoeia.

[0048] (1) Observation specifications: Under a standard laboratory environment of 25°C, pure β-NMN was placed in a white enamel dish and observed under natural light or a standard color temperature light source of 4000K. The β-NMN particles prepared in Example 3 were uniform and fine, with good looseness and flowability, and no visible mechanical impurities or moisture absorption and clumping. Through horizontal comparison with Sigma-Aldrich prototype standard and commercially available control products, the β-NMN prepared in Example 3 showed extremely high consistency in color saturation and texture, and no obvious sensory differences were observed.

[0049] (2) Solubility test: The β-NMN prepared in Example 3 is soluble in water to form a clear and transparent solution without turbidity or precipitation; it is slightly soluble in methanol, and the solution is slightly milky and does not have obvious stratification after standing; it is almost insoluble in non-polar solvents such as acetonitrile and ethyl acetate.

[0050] 4.2 Stability Evaluation Scheme The β-NMN sample prepared in Example 3 was placed in a brown glass bottle, forming a solid layer approximately 3 mm thick. The glass bottle was placed in a constant temperature and humidity chamber and subjected to two extreme conditions: high temperature (60±2℃, relative humidity 60%±5%) and high humidity (25±2℃, relative humidity 80%±5%). Samples were collected on days 0, 5, 10, 15, and 20, and their condition changes were observed to evaluate the stability of β-NMN under different storage conditions. The results are as follows: Figure 5 As shown.

[0051] Experimental observations show that environmental stress has a significant destructive effect on the physical appearance of products. Under thermal stress conditions, such as... Figure 5 As shown in (A), all samples exhibited a gradual color decay: the initial white powder turned pale yellow on day 5; subsequently, the color evolution path was light brown (day 10), dark brown (day 15) to grayish brown (day 20), indicating good tolerance. Figure 5 As shown in (B), humidity stress can maintain the white color within 20 days, thus better preserving the color of the product.

[0052] 4.3 Bioactivity and Isomer Specificity Testing Standards β-NMN, as a precursor of NAD⁺, plays a core role in anti-aging and metabolic regulation. Single-measure physicochemical assays are insufficient to fully characterize its biological efficacy. This study integrates enzymatic cascade reactions and fluorescence tracing technology to construct a function-guided bioactivity evaluation method. This method simulates the enzymatic conversion process of β-NMN in vivo, transforming it into a fluorescent derivative through a specific chemical reaction. The specific steps are as follows: 27.7 μL of 2 mol / L KOH and 27.7 μL of 20% (v / v) acetophenone solution are added to a centrifuge tube and equilibrated on ice for 2 min; then 125 μL of 88% formic acid is added, and the mixture is incubated at 37°C with shaking for 10 min; after the reaction, 240 μL of the reaction solution is transferred to a black 96-well microplate. Fluorescence intensity is measured using a Tecan multi-mode microplate reader at an excitation wavelength of 382 nm and an emission wavelength of 445 nm. All experiments were performed in triplicate to ensure statistical reliability of the data. The detection results for each sample are shown in Table 2.

[0053] Table 2. Comparison of fluorescence product intensities generated by β-NMN in the model enzymatic reaction. β-NMN sample Example 3 Commercially available raw material 1 Commercially available raw material 2 Commercially available raw material 3 Fluorescence intensity / au 120000 50000 48000 60000 Table 2 shows that, compared with commercially available top-grade β-NMN raw materials, the β-NMN prepared in the embodiments of the present invention exhibits extremely high bioefficacy, further proving that the β-NMN prepared in the embodiments of the present invention has the characteristics of high activity and high quality.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them; although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications can still be made to the specific implementation of the present invention or equivalent substitutions can be made to some technical features without departing from the spirit of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the technical solutions claimed in the present invention.

Claims

1. A multi-enzyme-based synthesis method for β-nicotinamide mononucleotide, comprising: PRPS enzyme solution, NAMPT enzyme solution, and Rbks enzyme solution were added to the substrate solution to form a β-NMN reaction system. This β-NMN reaction system was fermented for 200-550 min at 35-40 °C, pH 7.6-8.5, and 250-350 r / min. The substrate solution included nicotinamide, ribose-5-phosphate, and ATP. Based on final concentrations, the β-NMN reaction system comprised 0.5-3 mM nicotinamide, 0.5-3 mM ribose-5-phosphate, 10-30 mM ATP, 10-15 mM MgCl2, and 45-55 mM Tris-HCl buffer. The concentration ratio of nicotinamide phosphoribosyltransferase (NAMPT), ribokinase (Rbks), and phosphoribosyl pyrophosphate synthase (PRPS) in the β-NMN reaction system was 0.8-1.2:0.8-1.2: 0.8-1.2, the amino acid sequence of the nicotinamide phosphoribosyltransferase NAMPT is shown in SEQ ID NO. 1, and the ribokinase Rbks is derived from... Escherichia coli Rbks or source H. sapiens The amino acid sequence of the phosphoribosyl pyrophosphate synthase PRPS is shown in SEQ ID NO.

2.

2. The multi-enzyme-level synthesis method according to claim 1, characterized in that, The preparation method of the NAMPT enzyme solution includes: culturing the engineered strain BL21(DE3)-NAMPT expressing the nicotinamide phosphoribosyltransferase NAMPT in a culture medium at 36-38℃ and 130-150 rpm for 7-9 h; and then measuring the OD value. 600 When the concentration reaches the range of 0.6-0.8, cool to 15-17 ℃ and add IPTG at a concentration of 0.15-0.25 mM. Then, perform percolation expression for 14-18 h to obtain NAMPT fermentation broth. Centrifuge the NAMPT fermentation broth to collect the precipitate and obtain BL21(DE3)-NAMPT wet cells. First, add Tris-NaCl diluent to the BL21(DE3)-NAMPT wet cells and re-vortex. Then, add 0.9-1.1 mM PMSF for sonication to disrupt the cells. Centrifuge again at 11000-13000 rpm for 5-15 min and collect the supernatant to obtain a crude protein solution containing NAMPT. Elute the crude protein solution containing NAMPT with imidazole solution at 0℃-4℃ and dialyze to remove imidazole to obtain the NAMPT enzyme solution. The Tris-NaCl diluent contains 24-26 mM Tris and 145-155 mM NaCl.

3. The multi-enzyme-level synthesis method according to claim 1, characterized in that, The preparation method of the PRPS enzyme solution includes: constructing recombinant Escherichia coli BL21(DE3)-PRPS expressing the phosphoribosyl pyrophosphate synthase PRPS; placing the recombinant Escherichia coli BL21(DE3)-PRPS in resistant LB liquid medium and fermenting it under the conditions of 0.15-0.25 mmol / L total IPTG concentration, 35-40 ℃, and 150-250 r / min to obtain PRPS fermentation broth; centrifuging the PRPS fermentation broth to collect the precipitate and obtain BL21(DE3)-PRPS wet cells; washing the BL21(DE3)-PRPS wet cells with distilled water and adding buffer. BL21(DE3)-PRPS cells were suspended in buffer A; the recombinant E. coli BL21(DE3)-PRPS cells were disrupted using an ultrasonic cell disruptor under ice bath conditions to obtain a PRPS cell suspension; the PRPS cell suspension was subjected to freeze centrifugation, and the precipitate was discarded to obtain a crude protein solution containing PRPS; the crude protein solution of PRPS was purified by Ni affinity chromatography, wherein imidazole solution was used for elution, and the imidazole was removed by dialysis to obtain the PRPS enzyme solution; wherein the buffer A contained 19-21 mM imidazole, 24-26 mM Tris, and 140-160 mM NaCl.

4. The multi-enzyme-level synthesis method according to claim 1, characterized in that, The preparation method of the Rbks enzyme solution includes: constructing recombinant Escherichia coli BL21(DE3)-Rbks expressing the ribokinase Rbks; fermenting and culturing the recombinant Escherichia coli BL21(DE3)-Rbks, collecting the precipitate by centrifugation to obtain BL21(DE3)-Rbks wet cells; resuspending the BL21(DE3)-Rbks wet cells, sonicating and centrifuging to obtain the supernatant to obtain a crude protein solution containing Rbks; purifying the crude protein solution containing Rbks by column chromatography, wherein imidazole solution is used for elution, and imidazole is removed by dialysis to obtain the Rbks enzyme solution.

5. The multi-enzyme-level synthesis method according to claim 1, characterized in that, The fermentation time of the β-NMN reaction system is 200-300 min.

6. The method for synthesizing multiple enzymes according to any one of claims 1-5, characterized in that, Also includes: After the reaction of the β-NMN reaction system is completed, β-NMN fermentation broth is obtained; the β-NMN fermentation broth is separated, purified and dried to obtain pure β-NMN.

7. The multi-enzyme-level synthesis method according to claim 6, characterized in that, The separation and purification method of the β-NMN fermentation broth includes: separating and purifying the β-NMN fermentation broth using HPLC-UV method, wherein the HPLC detection conditions include: Agilent ZORBAX SB-C18 column; mobile phase A is phosphate, pH 5.5-6.0; mobile phase B is a mixture of methanol and formic acid, and the mass percentage of formic acid in the mixture is 0.008-0.012%; isocratic elution; elution time 5-20 min.

8. The multi-enzyme-level synthesis method according to claim 7, characterized in that, The drying method for the pure β-NMN product includes: drying the β-NMN purified by HPLC-UV method at 40-60 ℃ in a vacuum drying oven for 8-24 h.

9. The β-nicotinamide mononucleotide prepared by the multi-enzyme-level synthesis method according to any one of claims 1-8 has a purity ≥99.9% and a moisture content not higher than 1%.

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