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Method for preparing [beta]-nicotinamide mononucleotide

A single nucleotide and nicotinamide technology, applied in the field of biomedicine, can solve the problems of restricting the large-scale production of NMN in factories and high production costs, and achieve the effects of low cost, short production cycle and easy product purification.

Pending Publication Date: 2020-08-28
江苏易诺维生物医学研究院有限公司
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  • Description
  • Claims
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Problems solved by technology

[0006] Although the above method partially overcomes the defects of the chemical synthesis method, the relatively expensive nicotinamide ribose or nicotinamide, phosphoribose and ATP lead to high production costs, which limits the large-scale production of NMN in factories

Method used

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  • Method for preparing [beta]-nicotinamide mononucleotide
  • Method for preparing [beta]-nicotinamide mononucleotide
  • Method for preparing [beta]-nicotinamide mononucleotide

Examples

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Embodiment 1

[0033] Example 1 Cloning, expression and purification of β-nicotinamide adenine dinucleotide pyrophosphatase gene

[0034] (1) Cloning of β-nicotinamide adenine dinucleotide pyrophosphatase gene

[0035] The gene encoding β-nicotinamide adenine dinucleotide pyrophosphatase was from Escherichia coli BL21(DE3). The forward and reverse primers for PCR were designed to amplify the gene encoding β-nicotinamide adenine dinucleotide pyrophosphatase from the genomic DNA of Escherichia coli BL21(DE3).

[0036] The oligonucleotide sequence of the forward primer is:

[0037] 5'-GCCATGGCTGATATC GGATCC ATGGATCGTATAATTGAAAAATTAG-3', where the bases in italics (i.e. bases 16-21 from the 5' end) are enzyme cutting sites Bam HI;

[0038] The oligonucleotide sequence of the reverse primer is:

[0039] 5'-TTGTCGACGGAGCTC GAATTC TCACTCATACTCTGCCCG-3', where the bases in italics (i.e. the 16th-21st bases from the 5' end) are restriction sites Eco RI.

[0040] Extract genomic DNA from Esc...

Embodiment 2

[0048] Example 2 Enzymatic preparation of β-nicotinamide mononucleotide

[0049] (1) Add the pH 7.6 aqueous phase buffer (the aqueous phase buffer is Tris-Cl buffer with a concentration of 50 mM) into the reaction vessel, add the substrate β-nicotinamide adenine dinucleotide and Example 1 The prepared recombinant enzyme protein was mixed evenly to establish an in vitro reaction system. 1 L reaction containing 50 mM MgCl 2 , 5 mM β-nicotinamide adenine dinucleotide and 180 mg recombinant enzyme protein.

[0050] (2) The above reaction system was placed in a constant temperature shaker at 37°C for 30 min. After the reaction, the reaction solution was purified to obtain a purified reaction product. It was found that the conversion rate of β-nicotinamide adenine dinucleotide reached more than 90%, and the yield of β-nicotinamide mononucleotide was 1.5 g / L.

[0051] In the above step (1), recombinant cells containing β-nicotinamide adenine dinucleotide pyrophosphatase can also...

Embodiment 3

[0052] The detection of embodiment 3 reaction products

[0053] After the reaction, the pH of the reaction solution was adjusted to 3.0 with nitric acid, then 20 times the volume of acetone was added, mixed well, and placed at 4° C. overnight. Centrifuge at 4°C and 12000g for 10 min, collect the precipitate, and obtain a preliminary purified reaction product. Deionized water was used to dissolve the precipitate, and the reaction products were detected by thin layer chromatography (TLC method) and high performance liquid chromatography mass spectrometry (HPLC / MS method), and the analysis results were as follows: figure 2 and image 3 shown.

[0054] Wherein, the detection condition of TLC method is as follows:

[0055] The developer is a mixture of n-propanol, ethyl acetate and water, and the volume ratio of the three is 7:1:4.

[0056] The detection conditions of the HPLC / MS method are as follows:

[0057] Sample treatment before testing—dissolve the sample with deionize...

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Abstract

The invention puts forward a method for preparing [beta]-nicotinamide mononucleotide. In the method, [beta]-nicotinamide adenine dinucleotide is taken as a substrate, and reacts under a catalytic function of [beta]-nicotinamide adenine dinucleotide pyrophosphatase and / or a recombinant cell containing the [beta]-nicotinamide adenine dinucleotide pyrophosphatase to generate the [beta]-nicotinamidemononucleotide. Compared with an existing preparation method for the [beta]-nicotinamide mononucleotide, the method disclosed by the invention is simple to operate, is efficient, has a short production period, is environmentally-friendly, is low in cost and has an important application value in fields, including health care products, biological medicines and the like, and a product can be easily purified.

Description

technical field [0001] The invention relates to a method for preparing β-nicotinamide mononucleotide, in particular to a method for enzymatically synthesizing β-nicotinamide mononucleotide, which belongs to the technical field of biomedicine. Background technique [0002] According to reports, β-nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is the reaction product of nicotinamide phosphoribose transferase (NAMPT) and nicotinamide, etc. Nucleoside (Nicotinamide adenine dinucleotide, NAD+) salvage synthesis pathway key precursor. [0003] In recent years, studies have found that NMN has anti-aging (J Nutr Sci Vitaminol (Tokyo). 2016; 62(4): 272-276) and treatment of heart and brain ischemia (PLoS ONE. 2014; 9: e98972), Alzheimer Syndrome (BMCNeurol. 2015; 15: 19.), type 2 diabetes (Cell Metab. 2011; 14: 528-536), obesity and other pharmacological activities, and has broad application and development prospects in the fields of functional food and biomedicine. ...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12N15/55C12N15/70C12N9/14
CPCC12P19/30C12N9/14C12N15/70C12Y306/01022
Inventor 张新跃张智萍崔恒密
Owner 江苏易诺维生物医学研究院有限公司
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