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Method for quickly preparing [beta]-nicotinamide mononucleotide by enzymic method

A single nucleotide and nicotinamide technology, applied in the biological field, can solve problems such as poor prospects for industrial production, complicated NR chemical synthesis, and slow reaction speed, and achieve stable and rapid synthesis process, huge price advantage, and fast reaction speed Effect

Active Publication Date: 2021-04-13
ANHUI GSH BIO TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method seems simple, but the chemical synthesis of NR is also more complicated, with many pollutants, and the total cost is not lower than that of chemical synthesis of NMN, so the prospect of application in industrial production is not good.
The emerging enzymatic method mainly uses nicotinamide phosphoribosyltransferase (NAMPT, EC2.4.2.12) to catalyze phosphoribosyl pyrophosphate (PRPP) and nicotinamide (Nam) to generate NMN in the environment of ATP and magnesium ions. , but NAMPT has been proved to be a rate-limiting enzyme, which plays a role in controlling the reaction speed in the process of substance synthesis, and there are many substances that can inhibit it. Therefore, the reaction performed by this enzyme, the reaction The rate is relatively slow and the substrate conversion rate is relatively low

Method used

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  • Method for quickly preparing [beta]-nicotinamide mononucleotide by enzymic method
  • Method for quickly preparing [beta]-nicotinamide mononucleotide by enzymic method
  • Method for quickly preparing [beta]-nicotinamide mononucleotide by enzymic method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The preparation of embodiment 1 enzyme

[0032] All the enzymes in the method of the present invention can be obtained commercially, or are artificially modified enzymes with the same catalytic function.

[0033] The enzyme preparation process is as follows:

[0034] All enzyme numbers involved in this patent (including comparative examples) are EC 6.3.4.21, EC 2.4.2.19, EC 6.3.1.5, EC 2.4.2.57, EC 2.7.4.23, EC 2.4.2.7, EC 2.4.2.12, EC2.7.4.1, EC2.7.1.20, EC 2.7.4.3 and EC 2.7.4.B2.

[0035] Primers were designed according to the gene sequences of the enzymes used in the reaction, the gene fragments were amplified by PCR, and connected to the corresponding vectors (commercially available, single expression vector or multiple expression vectors could be selected), after the sequencing was correct, transfection Enter E.coliBL21 (DE3) strain (commercially available).

[0036] Insert the transformed E.coli BL21(DE3) monoclonal into LB medium, culture to the logarithmic p...

Embodiment 2

[0041] Embodiment 2 Nicotinic acid and PRPP are the reaction of substrate

[0042] The 50ml reaction system contains 0.15g nicotinic acid, 0.5g PRPP, 0.3g Tris, 0.2g potassium chloride, 0.25g magnesium chloride hexahydrate, 0.25g ammonium sulfate and 1.0g ATP, adjust the pH value to 7.0, add 500U EC 6.3 .4.21 enzyme and 500U EC6.3.1.5 enzyme to start the reaction. During the reaction, the pH value was controlled to be 7.0, and the temperature was 35°C.

[0043] After reacting for 1 hour, the generation amount of NMN detected by high performance liquid chromatography (HPLC) was 5.2g / L, and after 2 hours, it was detected that the generation amount of NMN was 6.9g / L, and after 3 hours, it was detected that the generation amount of NMN was 7.4g / L. Little to no increase. The conversion rate of niacin is more than 90%.

Embodiment 3

[0044] Embodiment 3 quinolinic acid and PRPP are the reaction of substrate

[0045] The 50ml reaction system contains 0.2g quinolinic acid, 0.5g PRPP, 0.3g Tris, 0.2g potassium chloride, 0.25g magnesium chloride hexahydrate, 0.25g ammonium sulfate and 1.0g ATP, adjust the pH value to 7.0, add 500U EC 2.4.2.19 Enzyme and 500U EC 6.3.1.5 Enzyme to start the reaction. During the reaction, the pH value was controlled to be 7.0, and the temperature was 35°C.

[0046] After reacting for 1 hour, the generation amount of NMN detected by high performance liquid chromatography (HPLC) was 4.6g / L. After 2 hours, the generation amount of NMN was detected as 6.3g / L. After 3 hours, the generation amount of NMN was detected as 6.7g / L. After that, NMN Little to no increase. The conversion rate of niacin is about 84%.

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Abstract

The invention discloses a method for quickly preparing [beta]-nicotinamide mononucleotide by an enzymic method. The method comprises the following steps of: taking PRPP as a raw material, and on the basis, adding nicotinic acid and / or quinolinic acid, ammonium ions, ATP and magnesium ions and / or manganese ions to form a reaction system; and under catalysis of an EC 6.3.4.21 enzyme and an EC 6.3.1.5 enzyme, or under the catalysis of an EC 2.4.2.19 enzyme and an EC 6.3.1.5 enzyme or under the catalysis of the EC 6.3.4.21 enzyme, the EC 6.3.1.5 enzyme and the EC 2.4.2.19 enzyme, enabling the reaction system to react to quickly prepare the [beta]-nicotinamide mononucleotide. Compared with a chemical method and a mixing method, the method disclosed by the invention has a stable and quick synthesis technology, no pollutants, including organic solvents and the like, are added in a whole process, and the method disclosed by the invention is environmentally friendly and is safe in production. Compared with other enzymic method production methods, the method disclosed by the invention has the advantages of a high reaction speed, a high conversion rate, a low production cost and a wide substrate range.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for rapidly preparing β-nicotinamide mononucleotide by enzymatic method. Background technique [0002] β-nicotinamide mononucleotide, referred to as NMN, has a molecular weight of 334.22. Studies have shown that it plays an important role in human cell repair and anti-aging. In recent years, journals such as Science, Nature, and Cell have published a number of articles on the efficacy of NMN. Harvard University experiments have confirmed that supplementing NMN can extend the lifespan of mammals by more than 30%. Therefore, NMN is regarded as an "elixir of life". , highly sought after by the health care products and pharmaceuticals market. However, the current market price of NMN is on the high side. The cheapest domestic product is about 1,500 yuan per 9,000 mg, and the price of imported products is higher, which cannot really benefit ordinary people. [0003] Existing NM...

Claims

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Application Information

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IPC IPC(8): C12P19/30
CPCC12P19/30C12N15/52C12N9/93C12N9/1077C12N9/1229C12Y603/04C12Y204/02019C12Y603/01005C12Y204/02007C12Y207/04023
Inventor 刘珊珊秦永发尹延明周稳文乔春鑫
Owner ANHUI GSH BIO TECH CO LTD
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