The non-specificity is 5apos; application of nucleotidase in preparation of nicotinamide ribose

Abstract

The invention provides application of non-specific 5 '-nucleotidase in production of beta-nicotinamide ribose (NR), and belongs to the technical field of biology. The invention discovers that the non-specific 5 '-nucleotidase derived from specific bacteria has obviously better catalytic activity for catalyzing the hydrolysis of nicotinamide nucleotide (NMN) to generate beta-nicotinamide ribose (NR) compared with UshA derived from escherichia coli, and the 5'-nucleotidase can be used for large-scale industrial production of beta-nicotinamide ribose.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and enzyme engineering, and specifically relates to the application of a non-specific 5'-nucleotidase derived from specific bacteria in the efficient catalytic hydrolysis of nicotinamide mononucleotide (NMN) to nicotinamide ribose (NR). Background Technology

[0002] β-Nicotinamide Riboside (NR) is a derivative of vitamin B3 (niacin). NR, along with nicotinamide (NAM) and nicotinamide mononucleotide (NMN), is considered to be nicotinamide adenine dinucleotide (NAD). + ) and nicotinamide adenine dinucleotide phosphate (NADP) + These are the precursors for the biosynthesis of two important coenzymes in the body. NAD + It plays a central role in human metabolic pathways and increases the level of NAD in the body. + Total NAD+ levels have been proven to be an effective strategy for treating various pathophysiological conditions. Extensive research data indicates that NR supplementation can effectively increase NAD+ levels in the body. + This level of activity can improve mitochondrial health, enhance metabolic function, and prevent cellular aging, among other potential health benefits. Therefore, NR has become one of the most widely studied NAD+ receptors in recent years. + One of the precursor molecules.

[0003] Natural nitrogen (NR) is present in very low amounts in natural substances and is mainly produced artificially. Currently, there are three main methods for producing NR: chemical synthesis, microbial fermentation, and enzyme-catalyzed synthesis.

[0004] Chemical synthesis typically uses nicotinamide (or ethyl nicotinate) or its pyridine N-(2,4-dinitrophenyl)onium salt and tetraacetylribose as raw materials. The main synthetic routes include: 1. Nicotinamide (NAM) and its derivatives undergo glycosylation with peracylated (halo)-D-ribose-furanose to form intermediates, thereby synthesizing nicotinamide ribose (NR) salts; 2. Nicotinamide ribose (NR) is formed by condensation reaction of N-(2,4-dinitrophenyl)-3-carbamoylpyridineonium salt with derivatives of D-furanose ribosamine. The main disadvantages of chemical synthesis include, for example, poor stereoselectivity leading to the production of byproducts such as α-nicotinamide ribose, high cost due to expensive activating reagents, and environmental pollution (Beilstein J. Org. Chem. 2019, 15, 401-430. CN107613990A, CN 110642897 A).

[0005]

[0006] Microbial fermentation, which involves constructing and reconstructing metabolic pathways through synthetic biology to directly accumulate NR within microorganisms, such as the synthesis of NR using E. coli fermentation as described in patent CN114854656B, offers a one-step product generation method. However, this method suffers from low yields, complex fermentation broth composition, and significant challenges in product separation.

[0007] Enzyme-catalyzed synthesis utilizes selective enzyme-catalyzed reactions to generate NR. Currently, there are two main types of enzyme reactions: 1. Taking CN116042748A as an example, NR is prepared using nicotinamide and purine nucleosides as substrates, employing a reversible substitution reaction between phosphate and bases catalyzed by nucleoside phosphorylase (NPase); 2. Taking CN120230814B as an example, NR is generated using nicotinamide mononucleotide as a substrate, catalyzed by a non-specific 5'-nucleotidylcholine hydrolase. Enzyme-catalyzed synthesis does not involve functional group protection-deprotection, activating reagents, or organic solvents, and has attracted much attention due to its environmental friendliness, high selectivity, and low cost.

[0008] However, existing enzyme-catalyzed synthesis methods still have shortcomings. For example, NPase-catalyzed conversion is an equilibrium reaction, making complete conversion difficult to achieve. Although hydrolysis reactions catalyzed by 5'-nucleotidases do not have equilibrium issues, their hydrolysis rates are limited, and enzyme expression and catalytic efficiency are low. As disclosed in CN113528562B, the non-specific 5'-nucleotidase UshA from *E. coli* has low expression levels, requiring a large amount of enzyme to ensure complete conversion, resulting in limited catalytic efficiency. CN120230814B discloses a 5'-nucleotidase U5 from *Shewanella* sp. HN-41. Although its catalytic activity is higher than UshA under high substrate concentration (40 g / L), it is believed that its catalytic activity is not effectively improved compared to UshA under low substrate concentration (5 g / L~20 g / L, especially below 20 g / L). Therefore, it is still difficult to meet the industrial production demand for high-efficiency, low-cost biocatalysts.

[0009] Therefore, there is still an urgent need in this field to discover and develop novel 5'-nucleotidases with higher catalytic activity in order to further reduce the production cost of NR and promote its industrial application. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a non-specific 5'-nucleotidase that is efficiently expressed in Escherichia coli and has significantly improved catalytic efficiency, and its application in the efficient catalytic hydrolysis of nicotinamide mononucleotide (NMN) to nicotinamide ribose (NR).

[0011] To achieve the above objectives, this invention utilizes bioinformatics analysis to mine 5'-nucleotidase resources. The 5'-nucleotidases were expressed using an *E. coli* expression system and then catalyzed using nicotinamide mononucleotide (NMN) as a substrate. The conversion efficiencies were screened and compared.

[0012] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0013] A first aspect of the present invention provides the use of a nonspecific 5'-nucleotidase in the preparation of nicotinamide ribose, wherein the nonspecific 5'-nucleotidase is selected from nonspecific 5'-nucleotidases derived from Citrobacter koseri, Kosakonia oryzendophytica, Klebsiella variicola, or Cedecea neteri.

[0014] Preferably, the nonspecific 5'-nucleotidase has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

[0015] More preferably, the nonspecific 5'-nucleotidase has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 5.

[0016] In some embodiments, the application is to prepare nicotinamide riboside (NR) by catalyzing the hydrolysis of nicotinamide mononucleotide (NMN) by the nonspecific 5'-nucleotidase.

[0017] A second aspect of the present invention provides a nucleic acid molecule encoding the nonspecific 5'-nucleotidase.

[0018] A third aspect of the present invention provides a method for preparing nicotinamide ribose (NR), comprising: under reaction conditions permissible by means of a nonspecific 5'-nucleotidase catalyzing the hydrolysis of nicotinamide mononucleotide (NMN) to generate nicotinamide ribose (NR).

[0019] In some embodiments, the nonspecific 5'-nucleotidase used in the above preparation method may be selected from crude enzyme solution, pure enzyme or immobilized enzyme, but is not limited thereto.

[0020] In some embodiments, the concentration of NMN in the reaction system can be from 5 g / L to 100 g / L, or further, from 10 g / L to 90 g / L or from 10 g / L to 50 g / L.

[0021] In some embodiments, the reaction temperature can be 25°C to 35°C, for example, 25°C to 30°C.

[0022] In some embodiments, the reaction system contains divalent metal ions, such as Mg²⁺.

[0023] In some embodiments, when the enzyme is used in the form of crude enzyme solution, the amount added to the reaction system is from 0.1 mL / L to 10 mL / L, and further, it can be from 0.2 mL / L to 5 mL / L or from 0.2 mL / L to 2 mL / L.

[0024] In some embodiments, the cells expressing the 5'-nucleotidase are Escherichia coli cells, for example, Escherichia coli BL21(DE3).

[0025] Beneficial effects

[0026] This invention is the first to verify that 5'-nucleotidases (SEQ ID NO: 2-5) derived from four bacteria, including Citrobacter korshinskii, exhibit highly efficient catalytic effects in the preparation of NMM to NR through hydrolysis. Under the same reaction conditions, the catalytic conversion rate (enzyme activity) of the 5'-nucleotidases of this invention is several times that of Escherichia coli UshA (SEQ ID NO: 1), specifically, 1.6 to 9.0 times that of Escherichia coli UshA, and preferably 4.5 to 9.0 times that of Escherichia coli UshA. For example, under 50 g / L NMN conditions, the 3-hour enzyme activity (269.7 U / mL) of the enzyme in SEQ ID NO: 2 of this invention is approximately 8.6 times that of UshA (31.2 U / mL), demonstrating a significant improvement in catalytic efficiency.

[0027] Meanwhile, compared to the 5'-nucleotidase U5 (CN120230814B) derived from Shewanella HN-41, which exhibits slightly enhanced catalytic activity in the prior art, U5 shows very limited catalytic effect at low substrate concentrations (5~20 g / L). Even at higher concentrations (40 g / L), the rate increase for UshA conversion (1.33-fold) is far lower than that of the 5'-nucleotidase of this invention. Therefore, the specific 5'-nucleotidase of this invention can exhibit more efficient catalytic activity over a wider range of substrate concentrations.

[0028] The 5'-nucleotidase provided by this invention can efficiently catalyze NMN substrates, significantly improving the synthesis efficiency of NR, solving the key technical bottleneck of slow hydrolysis rate in existing enzymatic routes, and providing a competitive and efficient biocatalyst for the large-scale industrial production of NR. Detailed Implementation

[0029] The present invention will become clearer to those skilled in the art through the following embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Materials and methods:

[0031] For experimental methods in the following examples where specific conditions are not specified, follow conventional methods and conditions, or select according to the product instructions.

[0032] Unless otherwise specified, the experimental procedures involved in gene cloning, molecular construction, cell culture, and protein expression shall be performed using conventional techniques in the field. In particular, the standard methods described in Molecular Cloning: A Laboratory Manual (Sambrook J, et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, 1989) or its subsequent versions may be referred to.

[0033] i) Reagents and instruments:

[0034] Unless otherwise specified, all reagents used in this invention are commercially available.

[0035] Nicotinamide mononucleotide (NMN) was purchased from Suzhou Yinhang Biotechnology Co., Ltd.; nicotinamide ribochloride (NR·Cl) was purchased from NET Chemical Research & Selection (Shanghai) Chemical Technology Co., Ltd.; and reagents such as 2-morpholine ethanesulfonic acid (MES) and magnesium chloride were purchased from Aladdin Reagent Co., Ltd.

[0036] ii) Vectors and strains:

[0037] The expression vector used was pET-30a(+) plasmid, purchased from Novagen; the host cell used was Escherichia coli BL21(DE3), purchased from Tiangen Biotech (Beijing) Co., Ltd.

[0038] iii) All genes were synthesized by optimizing codons using Universal Biosystems (Anhui) Co., Ltd., and then constructed into the vector pET-30a(+).

[0039] Example 1: Gene mining of 5'-nucleotidases, synthesis of expression plasmids, and construction of expression strains

[0040] 1. Gene mining for potential 5'-nucleotidases with high hydrolytic activity

[0041] To obtain a 5'-nucleotidase with high activity capable of catalyzing the hydrolysis of the substrate nicotinamide mononucleotide (NMN), the inventors used the reportedly high-activity 5'-nucleotidase UshA (SEQ ID NO: 1) from *Escherichia coli* as a reference, and conducted bioinformatics analysis and gene mining on genomic information from different microorganisms to screen for potential high-activity 5'-nucleotidases in the database. These 5'-nucleotidases are: an enzyme from *Citrobacter koseri* (SEQ ID NO: 2), an enzyme from *Kosakonia oryzendophytica* (SEQ ID NO: 3), an enzyme from *Klebsiella variicola* (SEQ ID NO: 4), and an enzyme from *Cedecea neteri* (SEQ ID NO: 5). Table 1 below shows the sequence identity of the above 5'-nucleotidases.

[0042] [Table 1]

[0043]

[0044] 2. Gene synthesis and expression plasmid construction

[0045] The four amino acid sequences (SEQ ID NO: 2~5) selected above, along with the control sequence (SEQ ID NO: 1, UshA), were sent to General Biosystems (Anhui) Co., Ltd. for gene sequence optimization and synthesis. The synthesized genes were cloned into the NdeI and HindIII restriction sites on the pET-30a(+) expression vector to obtain recombinant expression plasmids.

[0046] 3. Construction of expression strains

[0047] The correctly sequenced recombinant plasmids were transformed into E. coli BL21(DE3) competent cells, plated on LB plates containing kanamycin, and cultured overnight. Single clones were then picked to obtain the recombinant expression strain.

[0048] The sequencing work was completed by Suzhou Hongxun Biotechnology Co., Ltd.

[0049] Example 2: Expression of 5'-nucleotidase and preparation of crude enzyme solution

[0050] Each recombinant expression strain constructed in Example 1 was inoculated into LB liquid medium containing 50 mg / L kanamycin (50 mL medium in a 250 mL bottle, 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.0) and incubated overnight at 37°C with shaking. 1 mL of the culture was transferred to TB liquid medium (50 mL medium in a 250 mL bottle, 12 g / L peptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.31 g / L potassium dihydrogen phosphate, 12.54 g / L dipotassium hydrogen phosphate) and incubated at 37°C with shaking until OD... 600 The concentration was increased to 0.6-0.8. IPTG was added to a final concentration of 0.4 mM, and the mixture was incubated overnight at 25°C to induce protein expression.

[0051] After incubation, the culture medium (OD~10) was centrifuged at 4,000g for 10 min at 4°C, the supernatant was discarded, and the *E. coli* cells were collected. The collected *E. coli* cells were resuspended in pre-chilled 20 mL of pH 7.0 phosphate-buffered saline (PBS), and the *E. coli* cells were sonicated at 4°C. After cell lysis, the lysis buffer was centrifuged at 6,000g for 15 min at 4°C to remove the precipitate. The resulting supernatant was the crude enzyme solution containing recombinant 5'-nucleotidase. This crude enzyme solution can be directly used for subsequent catalytic reactions.

[0052] Example 3: Comparison of enzyme catalytic rates of different 5'-nucleotidases

[0053] To evaluate the catalytic reaction rate of the 5'-nucleotidase of the present invention, the following reaction catalyzing the hydrolysis of NMN to generate NR was carried out, and the concentration of substances in the conversion process was analyzed by high performance liquid chromatography (HPLC) to calculate the corresponding conversion rate.

[0054] Reaction system: The total volume of the reaction system is 20 mL, containing 50 mM MES buffer (pH 6.5) at a final concentration, 10 g / L NMN, 1 g / L MgCl2, and 0.2 mL / L crude enzyme solution in a shake flask.

[0055] The reaction was carried out at 30°C. Samples were taken at 0.5 hours and 1 hour of reaction, and the concentrations of NMN and NR were determined by HPLC. HPLC detection conditions followed standard methods in the art, and the conversion rate was calculated as follows.

[0056] Conversion rate (%) = NR molar concentration / (NR molar concentration + NMN molar concentration)

[0057] The experimental results are shown in Table 2 below.

[0058] [Table 2]

[0059]

[0060] As shown in Table 2 above, under the same enzyme dosage, the 5'-nucleotidases of SEQ ID NO: 2~5 of the present invention have a much faster reaction rate than UshA (SEQ ID NO: 1) from Escherichia coli in the process of catalyzing the generation of NR from NMN with a substrate concentration of 10 g / L.

[0061] Example 4: Comparison of the effects of different 5'-nucleotidases on the generation of NR from high concentrations of NMN

[0062] Reaction system: The total volume of the reaction system was 200 mL, containing 50 g / L NMN, 1 g / L MgCl2, and 2 mL / L crude enzyme solution in a shake flask. The reaction was stirred at 30 °C, and the pH was controlled at 6.5 during the reaction. Samples were taken at 3 hours and the conversion rate was detected by HPLC. The enzyme activity of the crude enzyme solution was calculated based on the conversion rate results, and the results are shown in Table 3 below.

[0063] [Table 3]

[0064]

[0065] Enzyme activity is defined as the catalytic ability to convert 1 μmol of substrate into product per minute. Taking the enzyme SEQ ID NO:1 in Example 4 as an example: after adding 2 mL / L of crude enzyme solution to a 50 g / L NMN solution, the conversion rate is 7.5% after 3 hours. Therefore, the enzyme activity in 1 L of the reaction system is... Therefore, the enzyme activity of the crude enzyme solution is 62.3U / 2mL = 31.2U / mL.

[0066] As shown in Table 3 above, under the same reaction conditions, the efficiency of the 5'-nucleotidase catalyzing substrate conversion of the present invention is significantly higher than that of Escherichia coli UshA, and can achieve rapid and efficient conversion of NMN to NR with a smaller amount of enzyme.

[0067] The above results demonstrate that the four specific 5'-nucleotidases (SEQ ID NO:2~5) provided by this invention exhibit significantly higher efficiency in catalyzing the hydrolysis of NMN to NR at a wide range of substrate concentrations, from low to high. This is in contrast to the control, *Escherichia coli* UshA (SEQ ID NO:1). The enzymes derived from *Citrobacter korshinskii* (SEQ ID NO:2) and *Cetis neculogens* (SEQ ID NO:5) show particularly outstanding performance, with catalytic activities (based on 3-hour conversion rates) 8.6 times and 5.8 times that of the control UshA, respectively. This indicates that the enzymes discovered in this invention have achieved a significant improvement in catalytic hydrolysis rate, effectively addressing the problem of insufficient enzyme catalytic efficiency in existing technologies, and possess broad prospects for industrial application.

[0068] The above description is merely an exemplary embodiment of the present invention and does not limit the scope of protection of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. The application of a nonspecific 5'-nucleotidase in the preparation of nicotinamide ribose, characterized in that, The nonspecific 5'-nucleotidase is selected from nonspecific 5'-nucleotidases derived from Citrobacter koseri, Kosakonia oryzendophytica, Klebsiella variicola, or Cedecea neteri.

2. The application according to claim 1, characterized in that, The nonspecific 5'-nucleotidase has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

3. The application according to claim 1, characterized in that, The application is to prepare nicotinamide ribose by catalyzing the hydrolysis of nicotinamide mononucleotides using the nonspecific 5'-nucleotidase.

4. A method for preparing nicotinamide ribose, characterized in that, The method comprises: under permissible reaction conditions, catalyzing the hydrolysis of nicotinamide mononucleotide by a nonspecific 5'-nucleotidase as described in claim 1 to generate nicotinamide ribose.

5. The method according to claim 4, characterized in that, The nonspecific 5'-nucleotidase is selected from crude enzyme solution, pure enzyme, or immobilized enzyme.

6. The method according to claim 4, characterized in that, In the reaction system of the reaction, the concentration of the nicotinamide mononucleotide is from 5 g / L to 100 g / L.

7. The method according to claim 5, characterized in that, The crude enzyme solution is added to the reaction system at a concentration of 0.1 mL / L to 10 mL / L.

8. The method according to claim 4, characterized in that, The cells expressing the 5'-nucleotidase are Escherichia coli cells.

9. The method according to claim 8, characterized in that, The Escherichia coli cells were Escherichia coli BL21(DE3).

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