Application of nonspecific 5'-nucleotidase in the preparation of nicotinamide riboside

Abstract

The present invention discloses the use of a nonspecific 5'-nucleotidase in the preparation of nicotinamide riboside. The nonspecific 5'-nucleotidase is selected from a nonspecific 5'-nucleotidase derived from Shewanella sp. HN-41. The nonspecific 5'-nucleotidase of the present invention can efficiently catalyze the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to produce nicotinamide riboside (NR), exhibits high enzymatic activity, and has value for industrial application.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme catalysis, and particularly relates to the application of a non-specific 5'-nucleotidase in the preparation of nicotinamide riboside. Background Art

[0002] Nicotinamide mononucleotide (β-Nicotinamide Mononucleotide, NMN) and Nicotinamide riboside (Nicotinamide riboside, NR) are both nicotinamide adenine dinucleotide (NAD + ), plays a core role in anti-aging, energy metabolism and DNA repair. However, there are significant differences in the bioavailability and metabolic pathways of nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR): ① Although nicotinamide mononucleotide (NMN) can directly increase the bioavailability of nicotinamide adenine dinucleotide (NAD + ) level, but its molecular weight is large and contains phosphate groups, making it difficult to penetrate the cell membrane. It needs to rely on specific transport proteins (such as Slc12a8) to enter the cell, resulting in low oral utilization. In addition, nicotinamide mononucleotide (NMN) is easily degraded into nicotinamide (NAM) in the digestive system, further reducing its effectiveness; ② Nicotinamide riboside (NR), as a smaller non-phosphorylated molecule, can efficiently pass through the cell membrane and is phosphorylated in the cell to generate nicotinamide mononucleotide (NMN), and finally synthesize nicotinamide adenine dinucleotide (NAD + ).

[0003] Nicotinamide adenine dinucleotide (NAD + ) is an oxidoreductase and other nicotinamide adenine dinucleotide (NAD + ) is an essential coenzyme for consuming enzymes, which not only plays an important role in maintaining the function of human cells but is also an important cofactor for many industrial biocatalytic processes. For most organisms, direct supplementation of nicotinamide adenine dinucleotide (NAD + ) is limited by two factors: nicotinamide adenine dinucleotide (NAD + ) is high cost and has very low assimilation efficiency. Therefore, supplementing nicotinamide adenine dinucleotide (NAD + ) precursor has become a major contributor to the production of nicotinamide adenine dinucleotide (NAD + ) level of effectiveness.

[0004] Nicotinamide riboside (NR) plays an important role in increasing the activity of nicotinamide adenine dinucleotide (NAD +) concentration is superior to conventional vitamin B3 (nicotinamide (NAM), niacin). Various biological effects of nicotinamide riboside (NR) have also been confirmed, such as enhancing oxidative metabolism, promoting the healing of difficult wounds, improving muscle mass and performance, improving inflammation and oxidative metabolism, improving gene silencing, and extending lifespan. In several human intervention studies, it was found that nicotinamide adenine dinucleotide (NAD + ) precursors can improve the + ) levels, with nicotinamide riboside (NR) showing the best effect. Furthermore, compared with other precursors, nicotinamide riboside (NR) has no side effects and is more suitable for applications in food, healthcare, and other fields.

[0005] Currently, there are two main chemical methods for the synthesis of commercial nicotinamide riboside (NR): 1. Nicotinamide (NAM) and its derivatives react with peracylated (halogen)-D-ribose-furanose to form intermediates through glycosylation reaction, and then synthesize nicotinamide riboside (NR) salt; 2. Nicotinamide riboside (NR) is formed by condensation reaction of N-(2,4-dinitrobenzene)-3-carbamoylpyridinium salt and D-ribofuranosamine derivatives.

[0006] The main drawback of chemically synthesized nicotinamide riboside (NR) (primarily using bromide and chloride salts) is that the glycosidic bond of NR salts is unstable compared to other nucleotides. Furthermore, the toxicity of bromide salts significantly limits their application, while chloride salts are easily hydrolyzed in aqueous solutions. Chemical synthesis methods are also relatively complex and expensive. Therefore, it is necessary to develop a method for the efficient synthesis of nicotinamide riboside (NR).

[0007] In 2022, the efficient synthesis of nicotinamide mononucleotide (NMN) from nicotinamide (NAM) was reported. In 2023, the use of the Escherichia coli-derived UshA protein to hydrolyze NMN led to the construction of a strain that efficiently produces nicotinamide riboside (NR). Currently reported plant-derived 5′-nucleotidases (such as BolN2, BolN5-X1, and BolN6 from kale) and the E. coli-derived UshA protein can catalyze the hydrolysis of NMN to nicotinamide riboside (NR), but their hydrolysis rates are limited, limiting the production of NR. Therefore, it is necessary to identify a 5′-nucleotidase that can efficiently catalyze the hydrolysis of high concentrations of NMN to nicotinamide riboside (NR). Summary of the Invention

[0008] The objective of this invention is to identify a 5′-nucleotidase that efficiently catalyzes the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to nicotinamide riboside (NR), thereby addressing existing technical challenges. Specifically, bioinformatics analysis and Yusong Bio's 5′-nucleotidase library were combined to explore and screen enzyme resources, resulting in the identification of a 5′-nucleotidase. After expression in an Escherichia coli expression system, the enzyme was catalyzed using nicotinamide mononucleotide (NMN) as a substrate. The conversion rates and selectivities of the wild-type and mutant enzymes were compared. The specific reaction scheme is shown below:

[0009]

[0010] The present invention solves the above technical problems through the following technical solutions.

[0011] The first aspect of the present invention provides a use of a non-specific 5'-nucleotidase in the preparation of nicotinamide riboside;

[0012] Wherein, the non-specific 5'-nucleotidase is selected from the non-specific 5'-nucleotidase derived from Shewanella sp. HN-41.

[0013] In some embodiments, the non-specific 5'-nucleotidase has the amino acid sequence shown in SEQ ID NO: 5.

[0014] In some specific embodiments, the amino acid sequence of the non-specific 5'-nucleotidase is shown in SEQ ID NO:5.

[0015] In some embodiments, the non-specific 5'-nucleotidase is encoded by a nucleotide sequence as shown in SEQ ID NO: 13.

[0016] In some specific embodiments, the nucleotide sequence encoding the non-specific 5'-nucleotidase is shown as SEQ ID NO: 13.

[0017] In some embodiments, the nicotinamide riboside is obtained by the non-specific 5'-nucleotidase-catalyzed reaction of nicotinamide mononucleotide.

[0018] A second aspect of the present invention provides a method for preparing nicotinamide riboside, the method comprising obtaining nicotinamide riboside by catalyzing a nicotinamide mononucleotide reaction with the non-specific 5'-nucleotidase;

[0019] The non-specific 5'-nucleotidase is selected from the non-specific 5'-nucleotidase derived from Shewanella sp. HN-41.

[0020] In some embodiments, the non-specific 5'-nucleotidase has the amino acid sequence shown in SEQ ID NO: 5.

[0021] In some specific embodiments, the amino acid sequence of the non-specific 5'-nucleotidase is shown in SEQ ID NO:5.

[0022] In some embodiments, the non-specific 5'-nucleotidase is encoded by a nucleotide sequence as shown in SEQ ID NO: 13.

[0023] In some specific embodiments, the nucleotide sequence encoding the non-specific 5'-nucleotidase is shown as SEQ ID NO: 13.

[0024] In some embodiments, the non-specific 5'-nucleotidase is used in a form selected from the group consisting of pure enzyme, crude enzyme solution, fermentation broth, enzyme powder, and immobilized enzyme.

[0025] In some embodiments, in the reaction system of the reaction: the concentration of the nicotinamide mononucleotide is at least 5 g / L.

[0026] In some preferred embodiments, the concentration of the nicotinamide mononucleotide is 5 g / L-40 g / L.

[0027] In some embodiments, the reaction system further comprises divalent metal ions.

[0028] In some preferred embodiments, the divalent metal ion is a magnesium ion.

[0029] In some embodiments, the reaction temperature of the reaction is 28-30°C.

[0030] In some embodiments, the crude enzyme solution is prepared by the following method: ultrasonically disrupting cells expressing the non-specific 5'-nucleotidase in a buffer solution, and collecting the supernatant after high-speed centrifugation.

[0031] In some embodiments, the cell expressing the non-specific 5'-nucleotidase is an Escherichia coli cell, for example, Escherichia coli BL21 (DE3).

[0032] In some preferred embodiments, the amount of the crude enzyme solution used in the reaction is 2-20% (v / v).

[0033] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0034] The reagents and raw materials used in the present invention are commercially available.

[0035] The positive progress effect of the present invention is:

[0036] The non-specific 5'-nucleotidase of the present invention can efficiently catalyze the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to produce nicotinamide riboside (NR), has high enzymatic activity and value for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is the result of the initial protein screening.

[0038] Figure 2 To test the expression and identification of UshA, U2 and U5 proteins.

[0039] Figure 3 Optimize and identify U5 protein expression.

[0040] Figure 4 To screen the protease activity under 20g / L conditions.

[0041] Figure 5 To screen the protease activity under 40g / L conditions.

[0042] Figure 6 This is the HPLC spectrum of nicotinamide mononucleotide (NMN) standard.

[0043] Figure 7 This is the HPLC spectrum of nicotinamide riboside (NR) standard. DETAILED DESCRIPTION

[0044] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0045] Since the currently reported 5′-nucleotidase activity cannot meet the needs, the inventors screened the enzyme library accumulated by Yusong Bio and various online databases, and screened out 7 enzymes from different sources that were marked as 5′-nucleotidase in the database. They were named U2, U3, U4, U5, U6, U7, and U8 respectively.

[0046] The HPLC detection method for nicotinamide mononucleotide (NMN) standard and nicotinamide riboside (NR) standard is shown in Table 1 below:

[0047] Table 1

[0048]

[0049] The test results of Nicotinamide Mononucleotide (NMN) Standard are as follows Figure 6As shown, the retention time is 2.488 min and the relative retention time is 0.79.

[0050] The test results of Nicotinamide Riboside (NR) Standard are as follows Figure 7 As shown, the retention time is 3.202 min and the relative retention time is 1.

[0051] Example 1: Genetic Screening for 5′-nucleotidase with High Catalytic Activity for Nicotinamide Mononucleotide (NMN)

[0052] 1.1 Genetic screening to obtain 5′-nucleotidases with high hydrolytic activity

[0053] In order to screen for 5′-nucleotidases with high enzymatic activity that can catalyze the hydrolysis of the substrate nicotinamide mononucleotide (NMN), the inventors mined the NCBI enzyme gene library based on the reported 5′-nucleotidase function of UshA protein (SEQ ID NO: 1), and mined the enzyme library accumulated by Yusong Biotechnology to select proteins annotated as 5′-nucleotidase in the database. They were named protein U2 from Vibrio cholerae (SEQ ID NO: 2), U3 from Streptococcus sp (SEQ ID NO: 3), U4 from Streptococcus infantis (SEQ ID NO: 4), U5 from Shewanella sp. HN-41 (SEQ ID NO: 5), U6 from Streptococcus sp. HSISS3 (SEQ ID NO: 6), U7 from Streptomyces sp. L-9-10 (SEQ ID NO: 7), and U8 from Staphylococcus aureus subsp. aureus CN1 (SEQ ID NO: 8). NO: 8).

[0054] The sequences are shown in Table 2:

[0055] Table 2 Screened amino acid sequences of 5′-nucleotidase

[0056]

[0057]

[0058]

[0059] The nucleotide sequences of the 5′-nucleotidases whose amino acid sequences are shown in SEQ ID NOs: 1-8 are shown in Table 3:

[0060] Table 3 Nucleotide sequence of UPO

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] 1.2 Transformation and expression of 5′-nucleotidase UshA and screening proteins

[0068] The selected amino acid sequences were optimized and synthesized by Jiangsu Saisuofei Biotechnology Co., Ltd. and cloned into the commercially available pCDFduet-1 plasmid. The synthesized plasmids were then transformed into cells engineered to inhibit nicotinamide riboside (NR) degradation (for transformation methods, see Huang Z et al., "Systematic engineering of Escherichiacoli for efficient production of nicotinamide riboside from nicotinamide and 3-cyanopyridine. Bioresour Technol. 2023 Jun;377:128953."). The corresponding engineered bacteria were then screened with antibiotics. The specific steps are as follows:

[0069] (1) Preparation of chemical transformation competent cells

[0070] Pipette 5 μL of laboratory-stored E. coli BL21 (DE3) glycerol strain with knockout of nicotinamide riboside (NR) degradation gene and streak it on LB medium (components shown in Table 4) to activate it. Pick a single activated colony and transfer it to LB medium. Incubate at 37°C in a shaking incubator until the OD 600 Competent cells were prepared at a concentration of 0.6. The preparation method was as follows: ice bath for 30 minutes; centrifugation to collect the cells; resuspend the cells in 15% glycerol; centrifugation to collect the cells; resuspend the collected cells in 0.1M CaCl2 and 15% glycerol, then aliquot and store in a -80°C ultra-low temperature freezer. All the above operations were performed in a low-temperature, sterile environment.

[0071] (2) Plasmid transformation

[0072] Take the competent cell and melt it on ice, add 2 μL of plasmid synthesized by Jiangsu Saisuofei Company, bathe in ice water for 30 minutes, heat shock at 42°C for 90 seconds, add 800 μL LB, culture at 37°C in a shaking incubator for 1 hour, centrifuge at 4000 rpm for 2 minutes, resuspend the precipitate and spread it on an LB plate containing spectinomycin, and culture at 37°C for 18 hours.

[0073] Table 4 LB medium formula

[0074]

[0075] (3) Inducible expression

[0076] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotics, and culture overnight in a shaker at 37°C and 220 rpm. Transfer the cultured bacteria into a triangular flask containing 10% of the volume of culture medium at a ratio of 1%, and culture in a shaker at 37°C and 220 rpm until the OD 600 When the pH value reaches 0.6-0.8, 0.5 mM IPTG is added and the cells are induced at 30°C and 220 rpm for 16 hours. After induction, the cells are collected by centrifugation.

[0077] 1.3 Activity Verification of 5′-nucleotidase UshA and Screened Proteins

[0078] Resuspend the cells in PBS buffer (pH 7.2) to maintain a consistent OD. Disrupt the resuspended cells using an ultrasonic sterilizer, and collect the supernatant by centrifugation. Add the supernatant to the reaction according to the ratios in Table 5. NMN was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The reaction components were mixed on ice, and the reaction was shaken at 30°C. Samples were collected at 0, 2, and 4 hours of reaction time. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was diluted 20-fold, filtered through a 0.22 μm filter, and analyzed for yield by HPLC.

[0079] Table 5 Reaction system for protein screening and activity verification

[0080]

[0081] The experimental results are shown in Figure 1 Under the substrate condition of 5 g / L, UshA, U2, and U5 can all effectively hydrolyze the substrate NMN.

[0082] Example 2: Detection of protein UshA, U2, and U5 expression

[0083] 2.1 Screening protein expression test

[0084] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotics, and culture it in a shaker at 37°C and 220 rpm overnight. Transfer the cultured bacterial solution to a triangular flask containing 10% volume culture medium at a ratio of 1%, and culture it in a shaker at 37°C and 220 rpm until the OD 600 When the pH value reaches 0.6-0.8, 0.5 mM IPTG is added and the cells are induced at 30°C and 220 rpm for 16 hours. After induction, the cells are collected by centrifugation.

[0085] The collected cells were resuspended in PBS buffer to maintain a consistent OD. The resuspended cells were disrupted using an ultrasonic disruptor with the following program: 6-amplitude, 1-step, 2s on, 3s off, 40% power, 20 min. After disruption, the cells were centrifuged at 10,000 g for 20 min and expression was determined by SDS-PAGE (see Figure 2 The results showed that UshA and U2 were expressed normally, while U5 had no obvious band. The supernatant was used as crude enzyme solution and stored at 4°C.

[0086] 2.2 Optimization of screening protein U5 expression

[0087] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotics, and culture it in a shaker at 37°C and 220 rpm overnight. Transfer the cultured bacterial solution to a triangular flask containing 10% volume culture medium at a ratio of 1%, and culture it in a shaker at 37°C and 220 rpm until the OD 600 When the pH value reaches 0.6-0.8, add 0.5 mM IPTG and induce at 16°C, 220 rpm for 20 hours. After induction, collect the cells by centrifugation.

[0088] The collected bacteria were resuspended in PBS buffer (pH 7.2) and disrupted using an ultrasonic disruptor. The disruption program was: 6 horn, project number 1, on for 2 seconds, off for 3 seconds, 40% power, and 20 minutes. After disruption, the cells were centrifuged at 10,000 g for 20 minutes and expression was determined by SDS-PAGE (see Figure 3 The results showed that when induced at 16°C, U5 was expressed in large quantities as inclusion bodies.

[0089] Example 3: Optimization of reaction conditions

[0090] 3.1 20 g / L substrate reaction

[0091] In this example, the enzyme catalyzed reaction was carried out on the screened enzymes using the reaction system shown in the following table at 20 g / L substrate.

[0092] Table 6 Screening protein catalytic reaction system

[0093]

[0094] The crude enzyme solution was added to the reaction according to the ratio in Table 5. The reaction components were mixed on ice and the reaction system was shaken at 30°C. Samples were taken at 0, 15, 30, 45, 60, 60, 90, 120, 150, and 180 minutes. After the reaction was terminated by adding hydrochloric acid to adjust the pH to 3, the samples were centrifuged at 12,000 rpm for 10 minutes. The supernatant was diluted 20 times and filtered through a 0.22 μm filter membrane. The yield was analyzed by HPLC. The results are shown in Table 5. Figure 4 .

[0095] Under the condition of substrate concentration of 20 g / L, both UshA and U5 can efficiently convert the substrate. The reaction rate of U5 is higher than that of UshA before 150 min, and most of the substrate is converted within 150 min; after 150 min, the reaction rates of U5 and UshA are close.

[0096] 3.2 40 g / L substrate reaction

[0097] In order to compare the reaction rate of the screened enzymes when the substrate is sufficient, the substrate concentration was increased to 40 g / L for the enzyme-catalyzed reaction. The reaction system is shown in Table 7. Samples were taken at 0, 20, 35, 50, 65, 90, and 120 min. The samples were added with hydrochloric acid to pH 3 to terminate the reaction. The samples were centrifuged at 12,000 rpm for 10 min. The supernatant was diluted 20 times and filtered through a 0.22 μm filter membrane. The yield was analyzed by HPLC. Figure 5 .

[0098] Table 7 Screening protein catalytic reaction system

[0099]

[0100] Under the substrate condition of 40 g / L substrate, the substrate conversion rate of U5 was 6.35 g / L / h, which was significantly higher than that of UshA (4.785 g / L / h).

[0101] In summary, the present invention screened the NCBI database and the Pueraria lobata protein database based on enzyme function requirements, resulting in a highly active 5′-nucleotidase, U5. Compared to the previously reported 5′-nucleotidase, UshA, U5 exhibits high activity at low protein expression levels.

Claims

1. Use of a non-specific 5'-nucleotidase in the preparation of nicotinamide riboside; in, The non-specific 5'-nucleotidase is selected from the group consisting of Shewanella HN-41 ( Shewanella sp. HN-41); the amino acid sequence of the nonspecific 5'-nucleotidase is shown in SEQ ID NO:

5.

2. The use according to claim 1, characterized in that The non-specific 5'-nucleotidase is encoded by the nucleotide sequence shown in SEQ ID NO:

13.

3. The use according to claim 2, characterized in that The nicotinamide riboside is obtained by the non-specific 5'-nucleotidase catalyzing the reaction of nicotinamide mononucleotide.

4. A method for preparing nicotinamide riboside, characterized in that: The method comprises the steps of obtaining nicotinamide riboside by catalyzing a nicotinamide mononucleotide reaction with a non-specific 5'-nucleotidase; The non-specific 5'-nucleotidase is selected from the group consisting of Shewanella HN-41 ( Shewanella sp. HN-41); the amino acid sequence of the nonspecific 5'-nucleotidase is shown in SEQ ID NO:

5.

5. The method according to claim 4, wherein The non-specific 5'-nucleotidase is used in a form selected from pure enzyme, crude enzyme solution, fermentation broth, enzyme powder and immobilized enzyme.

6. The method according to claim 4, wherein In the reaction system of the reaction: the concentration of the nicotinamide mononucleotide is at least 5 g / L; And / or, the reaction system further comprises divalent metal ions; And / or, the reaction temperature of the reaction is 28-30°C.

7. The method according to claim 6, wherein The concentration of the nicotinamide mononucleotide is 5g / L-40g / L; And / or, the divalent metal ion is a magnesium ion.

8. The method according to claim 5, wherein The crude enzyme solution is prepared by the following method: cells expressing the non-specific 5'-nucleotidase are ultrasonically disrupted in a buffer solution, and the supernatant is collected after high-speed centrifugation.

9. The method according to claim 8, wherein The amount of the crude enzyme solution used in the reaction is 2-20% (v / v); And / or, the cell expressing the non-specific 5'-nucleotidase is an Escherichia coli cell.

10. The method according to claim 9, wherein The Escherichia coli cell is Escherichia coli BL21 (DE3).

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