Application of non-specific 5 '-nucleotidase in preparation of nicotinamide ribose

Abstract

The invention discloses an application of non-specific 5 '-nucleotidase in preparation of nicotinamide ribose. The non-specific 5 '-nucleotidase is selected from non-specific 5'-nucleotidase from shewanella sp.HN-41. The non-specific 5 '-nucleotidase disclosed by the invention can be used for efficiently catalyzing high-concentration nicotinamide mononucleotide (NMN) to be hydrolyzed to generate nicotinamide ribose (NR), and has relatively high enzyme activity and industrial application value.

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

[0003] Nicotinamide adenine dinucleotide (NAD + ) is an essential coenzyme for oxidoreductases and other nicotinamide adenine dinucleotide (NAD + )-consuming enzymes. It not only plays an important role in maintaining the functions of human cells, but also is an important cofactor for many industrial biocatalytic processes. For most organisms, the exogenous supplementation of nicotinamide adenine dinucleotide (NAD + ) is limited by two factors: the high cost of nicotinamide adenine dinucleotide (NAD + ) and its very low assimilation efficiency. Therefore, supplementing the precursor of nicotinamide adenine dinucleotide (NAD + ) has become an effective method to increase the level of nicotinamide adenine dinucleotide (NAD + ).

[0004] Nicotinamide riboside (NR) in increasing nicotinamide adenine dinucleotide (NAD +has an advantage over conventional vitamin B3 (nicotinamide (NAM), niacin) in terms of concentration. Various biological functions of nicotinamide riboside (NR) have also been confirmed, such as enhancing oxidative metabolism, promoting the healing of refractory 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 dietary supplements of nicotinamide adenine dinucleotide (NAD + )precursors can improve the level of nicotinamide adenine dinucleotide (NAD + ), among which nicotinamide riboside (NR) has the best effect. In addition, 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 mainly two chemical methods for synthesizing commercially available nicotinamide riboside (NR): 1. Nicotinamide (NAM) and its derivatives react with peracylated (halo)-D-ribofuranose through glycosylation reaction to form an intermediate, and then synthesize nicotinamide riboside (NR) salt; 2. Using N-(2,4-dinitrophenyl)-3-carbamoylpyridinium salt and derivatives of D-ribofuranamine to form nicotinamide riboside (NR) through condensation reaction.

[0006] The main disadvantage of chemically synthesized nicotinamide riboside (NR) (mainly including bromide salt and chloride salt) is that the glycosidic bond of nicotinamide riboside (NR) salt is unstable compared with other nucleotides. Moreover, the bromide salt is toxic, greatly limiting its application; the chloride salt is prone to hydrolysis in aqueous solution. The chemical synthesis method is also relatively complex and expensive. Therefore, it is necessary to develop a method that can effectively synthesize nicotinamide riboside (NR).

[0007] In 2022, it was reported that the efficient synthesis of nicotinamide mononucleotide (NMN) from nicotinamide (NAM) was achieved. In 2023, it was reported that the UshA protein derived from Escherichia coli was used to hydrolyze nicotinamide mononucleotide (NMN) to construct a strain that can efficiently generate nicotinamide riboside (NR). Currently reported plant-derived 5'-nucleotidases (such as BolN2, BolN5-X1, and BolN6 in kale) and the UshA protein derived from Escherichia coli can catalyze the hydrolysis of nicotinamide mononucleotide (NMN) to generate nicotinamide riboside (NR), but their hydrolysis rate is limited, restricting the generation of nicotinamide riboside (NR). Therefore, it is necessary to find a 5'-nucleotidase that can efficiently catalyze the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to generate nicotinamide riboside (NR). Summary of the Invention

[0008] The object of the present invention is to find a 5'-nucleotidase that can efficiently catalyze the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to produce nicotinamide ribose (NR) to solve the problems in the prior art. Specifically, in combination with bioinformatics analysis and the 5'-nucleotidase library of Yusong Biology, enzyme resources are mined and screened to obtain 5'-nucleotidase. After expression in the Escherichia coli expression system, a catalytic reaction is carried out using nicotinamide mononucleotide (NMN) as a substrate, and the conversion rate and selectivity of the wild type and mutants are compared. The specific reaction route is as follows:

[0009]

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

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

[0012] Among them, 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 an amino acid sequence as shown in SEQ ID NO: 5.

[0014] In some specific embodiments, the amino acid sequence of the non-specific 5'-nucleotidase is as 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 as shown in SEQ ID NO: 13.

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

[0018] The second aspect of the present invention provides a method for preparing nicotinamide ribose, the method comprising obtaining nicotinamide ribose by catalyzing the reaction of nicotinamide mononucleotide 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 as shown in SEQ ID NO: 5.

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

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

[0024] In some embodiments, the form of use of the non-specific 5'-nucleotidase is selected from 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 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: sonicating the cells expressing the non-specific 5'-nucleotidase in a buffer, and taking the supernatant after high-speed centrifugation.

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

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

[0033] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

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

[0035] The positive and progressive effects of the present invention are as follows:

[0036] The non-specific 5'-nucleotidase of the present invention can efficiently catalyze the hydrolysis of high-concentration nicotinamide mononucleotide (NMN) to generate nicotinamide riboside (NR), and has high enzyme activity and the value of industrial application. Brief Description of the Drawings

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

[0038] Figure 2 It is the small-scale expression identification of UshA, U2, and U5 proteins.

[0039] Figure 3 It is the expression optimization identification of U5 protein.

[0040] Figure 4 It is the protease activity screening and identification under the condition of 20 g / L.

[0041] Figure 5 It is the protease activity screening and identification under the condition of 40 g / L.

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

[0043] Figure 7 It is the HPLC chromatogram of nicotinamide riboside (NR) standard. Detailed Embodiments

[0044] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods without specific conditions in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0045] Since the activities of the reported 5'-nucleotidases currently cannot meet the requirements, the inventor screened the enzyme libraries accumulated by Yusong Biology and various online databases, and screened out 7 enzymes from different sources that are labeled as 5'-nucleotidases in the database, and named them U2, U3, U4, U5, U6, U7, and U8 respectively.

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

[0047] Table 1

[0048]

[0049] The detection results of nicotinamide mononucleotide (NMN) standard are as Figure 6As shown, the retention time is 2.488 min and the relative retention time is 0.79.

[0050] The detection results of nicotinamide riboside (NR) standard are as Figure 7 shown, the retention time is 3.202 min and the relative retention time is 1.

[0051] Example 1: Gene Screening of 5'-Nucleotidase with High Catalytic Activity towards Nicotinamide Mononucleotide (NMN)

[0052] 1.1 Gene Screening to Obtain 5'-Nucleotidase with High Hydrolysis Activity

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

[0054] The sequences are shown in Table 2:

[0055] Table 2 Amino Acid Sequences of 5'-Nucleotidase Obtained by Screening

[0056]

[0057]

[0058]

[0059] The nucleotide sequences of 5'-nucleotidase with amino acid sequences as shown in SEQ ID NO: 1-8 are shown in Table 3:

[0060] Table 3 Nucleotide Sequences of UPO

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067] 1.2 Transformation and expression of 5'-nucleotidase UshA and screening protein

[0068] The screened amino acid sequences were sent to Jiangsu Saisuofei Biotechnology Co., Ltd. for gene sequence optimization and synthesis, and cloned onto the commercial plasmid pCDFduet-1. The synthesized plasmids were respectively transformed into the modified chassis cells resistant to nicotinamide riboside (NR) degradation (for the transformation method, see Huang Z et al., Systematic engineering of Escherichia coli for efficient production of nicotinamide riboside from nicotinamide and 3-cyanopyridine. Bioresour Technol. 2023 Jun;377:128953.). After antibiotic screening, the corresponding engineered bacteria were obtained. The specific operation steps are as follows:

[0069] (1)Preparation of chemically competent cells

[0070] Take 5 μL of the glycerol stock of Escherichia coli BL21(DE3) with the nicotinamide riboside (NR) degradation gene knocked out, which is stored in the laboratory, and streak it on an LB medium (the components are shown in Table 4) plate for activation. Pick the activated single colony into the LB medium and culture it in a shaker at 37 °C until the OD 600 : When it reaches 0.6, prepare the competent cells. The preparation method is as follows: Ice bath for 30 min; Centrifuge to collect the bacteria; Resuspend the bacteria with 15% glycerol; Centrifuge to collect the bacteria again; Resuspend the collected bacteria with 0.1 M CaCl2 and 15% glycerol, then aliquot and store in an ultra-low temperature freezer at -80 °C. All the above operations are carried out in a low-temperature and sterile environment.

[0071] (2)Plasmid transformation

[0072] Take the competent cells and let them thaw on ice. Add 2 μL of the plasmid synthesized by Jiangsu Saisuofei Company, and incubate in an ice-water bath for 30 min. After heat shock at 42 °C for 90 s, add 800 μL of LB, and culture in a shaker at 37 °C for 1 h. After the culture is completed, centrifuge at 4000 rpm for 2 min. Take the precipitate, resuspend it, and spread it on an LB plate containing spectinomycin, and incubate at a constant temperature of 37 °C for 18 h.

[0073] Table 4 LB Medium Formula

[0074]

[0075] (3)Induced Expression

[0076] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotic, and place it in a shaker at 37 °C and 220 rpm for overnight culture. Transfer the cultured bacterial solution to a triangular flask containing 10% (v / v) medium at a ratio of 1%, and culture in a shaker at 37 °C and 220 rpm until the OD 600 reaches between 0.6 - 0.8, then add 0.5 mM IPTG, and induce at 30 °C and 220 rpm for a total of 16 h. After the induction is completed, centrifuge to collect the bacterial cells.

[0077] 1.3 Activity Verification of 5'-Nucleotidase UshA and Screening Protein

[0078] Resuspend the bacterial cells using PBS buffer (pH 7.2) to control the same OD of the resuspended bacterial cells. Use an ultrasonic cell disruptor to break the resuspended bacterial cells, and centrifuge to collect the supernatant of the disrupted cells. Add the reaction components to the centrifuged supernatant according to the ratio in Table 5. NMN is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. Mix the reaction components on ice, and place the mixed reaction system in a shaker at 30 °C. Take samples at 0, 2, and 4 h of the reaction. After centrifuging at 12000 rpm for 10 min, dilute the supernatant 20 times, filter it through a 0.22 μm filter membrane, and analyze its yield by HPLC.

[0079] Table 5 Reaction System for Activity Verification of Screening Protein

[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: Expression Detection of Proteins UshA, U2, and U5

[0083] 2.1 Small-Scale Experiment on Screening Protein Expression

[0084] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotic, and place it in a shaker at 37°C and 220 rpm for overnight culture. Transfer the cultured bacterial solution to a triangular flask containing 10% (v / v) medium at a ratio of 1%, and culture it in a shaker at 37°C and 220 rpm until the OD 600 reaches between 0.6 and 0.8, then add 0.5 mM IPTG, and induce at 30°C and 220 rpm for a total of 16 h. After the induction is completed, centrifuge to collect the bacterial cells.

[0085] The collected bacterial cells are resuspended with PBS buffer to control the same OD. The resuspended bacterial solution is broken by an ultrasonic crusher, and the breaking program is: amplitude transformer 6, project number 1, on for 2 s and off for 3 s, 40% power, working for 20 min. After breaking the bacteria, centrifuge at 10000g for 20 min and perform SDS-PAGE to detect the expression (see Figure 2 ). The results show that UshA and U2 are normally expressed, and there is no obvious band for U5. The centrifuged supernatant is used as the crude enzyme solution and stored at 4°C.

[0086] 2.2 Screening for optimization of protein U5 expression

[0087] Pick a single colony from the transformation plate into an LB test tube, add the corresponding antibiotic, and place it in a shaker at 37°C and 220 rpm for overnight culture. Transfer the cultured bacterial solution to a triangular flask containing 10% (v / v) medium at a ratio of 1%, and culture it in a shaker at 37°C and 220 rpm until the OD 600 reaches between 0.6 and 0.8, then add 0.5 mM IPTG, and induce at 16°C and 220 rpm for a total of 20 h. After the induction is completed, centrifuge to collect the bacterial cells.

[0088] The collected bacterial cells are resuspended with PBS buffer (pH 7.2). The resuspended bacterial solution is broken by an ultrasonic crusher, and the breaking program is: amplitude transformer 6, project number 1, on for 2 s and off for 3 s, 40% power, working for 20 min. After breaking the bacteria, centrifuge at 10000g for 20 min and perform SDS-PAGE to detect the expression (see Figure 3 ). The results show that when induced at 16°C, a large amount of U5 is expressed as inclusion bodies.

[0089] Example 3: Optimization of reaction conditions

[0090] 3.1 Reaction with 20 g / L substrate

[0091] In this example, using the following reaction system, under the condition of 20 g / L substrate, the screened enzyme is subjected to an enzymatic reaction. The reaction system is shown in Table 6:

[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 components of the reaction were mixed on ice, and the mixed reaction system was carried out on a shaker at 30 °C. Samples were taken at 0, 15, 30, 45, 60, 60, 90, 120, 150, and 180 min of the reaction. After adding hydrochloric acid to adjust the pH of the sample to 3 to terminate the reaction, it was centrifuged at 12000 rpm for 10 min. The supernatant was diluted 20 times and then filtered through a 0.22 μm filter membrane and analyzed by HPLC for its yield. The results are shown in Figure 4 。

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

[0096] 3.2 Reaction with a substrate of 40 g / L

[0097] To compare the reaction rates of the screened enzymes when the substrate was 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 of the reaction. After adding hydrochloric acid to adjust the pH of the sample to 3 to terminate the reaction, it was centrifuged at 12000 rpm for 10 min. The supernatant was diluted 20 times and then filtered through a 0.22 μm filter membrane and analyzed by HPLC for its yield. The results Figure 5 。

[0098] Table 7 Reaction system for screening protein catalysis

[0099]

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

[0101] In summary, based on the requirements of enzyme function, the present invention screened in the NCBI database and the Yusong protein database to obtain a 5'-nucleotidase U5 with high activity. Compared with the reported 5'-nucleotidase UshA, U5 can exhibit high activity with a low protein expression level.

Claims

1. Use of a non-specific 5'-nucleotidase in the preparation of nicotinamide riboside; Among them, The non-specific 5'-nucleotidase is selected from the non-specific 5'-nucleotidase derived from Shewanella sp. HN-41.

2. The application according to claim 1, characterized in that The non-specific 5'-nucleotidase has an amino acid sequence as shown in SEQ ID NO:

5.

3. The application according to claim 1, wherein The non-specific 5'-nucleotidase is encoded by a nucleotide sequence as shown in SEQ ID NO:

13.

4. The application according to claim 3, wherein The nicotinamide riboside is obtained by catalyzing the reaction of nicotinamide mononucleotide with the non-specific 5'-nucleotidase.

5. A method for preparing nicotinamide riboside, characterized in that, The method includes obtaining nicotinamide riboside by catalyzing the reaction of nicotinamide mononucleotide with a non-specific 5'-nucleotidase; The non-specific 5'-nucleotidase is selected from the non-specific 5'-nucleotidase derived from Shewanella sp. HN-41.

6. The method according to claim 5, characterized in that The non-specific 5'-nucleotidase has an amino acid sequence as shown in SEQ ID NO:

5.

7. The method according to claim 6, wherein The use form of the non-specific 5'-nucleotidase is selected from pure enzyme, crude enzyme solution, fermentation broth, enzyme powder and immobilized enzyme.

8. The method according to claim 5, 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 contains divalent metal ions; and / or, the reaction temperature of the reaction is 28-30 °C.

9. The method according to claim 8, wherein The concentration of the nicotinamide mononucleotide is 5 g / L - 40 g / L; and / or, the divalent metal ion is magnesium ion.

10. The method according to any one of claims 7-9, characterized in that, The crude enzyme solution is prepared by the following method: sonicating the cells expressing the non-specific 5'-nucleotidase in a buffer solution, and taking the supernatant after high-speed centrifugation.

11. The method according to claim 10, wherein The dosage of the crude enzyme solution in the reaction is 2-20% (v / v); and / or, the cells expressing the non-specific 5'-nucleotidase are Escherichia coli cells.

12. The method according to claim 11, wherein The Escherichia coli cells are Escherichia coli BL21(DE3).

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