Enzymatic synthesis method of nicotinamide mononucleotide

An enzymatic synthesis, single nucleotide technology, applied in the direction of fermentation, etc., can solve the problems of high price, unfavorable enzymatic NMN industrialization, and high difficulty in separation of products and raw materials

Active Publication Date: 2021-06-15
深圳希吉亚生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymatic production of NMN is a green and environmentally friendly biocatalytic method. Most of the reported enzymatic production of NMN uses ATP as a reaction raw material. However, on the one hand, the high price of ATP leads to hi...

Method used

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  • Enzymatic synthesis method of nicotinamide mononucleotide

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Embodiment 1

[0050] Enzyme preparation

[0051] According to the sequences of the following four enzyme genes of cytidylate hydrolase (phNn), phosphoribosyl pyrophosphorylase (rpPk), nicotinamide phosphoribosyltransferase (NAMPT) and polyphosphate kinase (PPK), the art A well-known design method uses primer design software to design the amplification primer pairs respectively.

[0052] Extracting Shigella sonnei (S.sonnei) genomic DNA, using it as a template, using the phNn amplification primer pair to amplify the cytidylate hydrolase fragment of Shigella sonnei, and connecting it to the pET24a vector;

[0053] Extract Pyrococcus (P.horikoshii) genomic DNA, use it as a template, utilize the rpPk amplification primer pair to amplify the phosphoribosyl pyrophosphorylase fragment of Pyrococcus, and connect it to the pET24a vector;

[0054] Extract Haemophilus ducreyi (H.ducreyi) genomic DNA, use it as a template, use the NAMPT amplification primer pair to amplify the nicotinamide phosphoribo...

Embodiment 2

[0073] This embodiment provides an enzymatic synthesis method of nicotinamide mononucleotide, and the specific reactions that occur during the reaction process of the enzymatic synthesis method are as follows:

[0074]

[0075] The enzymatic synthesis method comprises the following steps:

[0076] S0: Add cytidylic acid with a final concentration of 100mM in 1L of pure water, adjust the pH to 7.5 with 40% sodium hydroxide solution, add 500 U of cytidylic acid hydrolase, stir at 35°C for 2 hours, and heat to 80°C , cooled and centrifuged to obtain a ribose-5-phosphate solution.

[0077] S1: Add nicotinamide and 50 mM MgCl to the ribose-5-phosphate solution obtained in S0 with a final concentration of 100 mM 2 , 60mM sodium hexametaphosphate and 4mM cytidylic acid, adjust the pH to 7.5 with 40% sodium hydroxide solution, add 1000U of phosphoribosyl pyrophosphorylase, 1000U of polyphosphate kinase and 500U of nicotinamide phosphoribosyl Transferase, stirred at 35°C for 6 hou...

Embodiment 3

[0081] This embodiment provides an enzymatic synthesis method of nicotinamide mononucleotide, comprising the following steps:

[0082] S0: Add cytidylic acid with a final concentration of 100mM in 1L of pure water, adjust the pH to 7.5 with 40% sodium hydroxide solution, add 500 U of cytidylic acid hydrolase, stir at 35°C for 2 hours, and heat to 80°C , cooled and centrifuged to obtain a ribose-5-phosphate solution.

[0083] S1: In the ribose-5-phosphate solution obtained in S0, add nicotinamide with a final concentration of 100 mM, MgCl with a final concentration of 50 mM, sodium hexametaphosphate with a final concentration of 60 mM and cytidylic acid with a final concentration of 4 mM, and use 40 % sodium hydroxide solution to adjust the pH to 7.5, add 1500 U of phosphoribosyl pyrophosphorylase, 1500 U of polyphosphate kinase and 500 U of nicotinamide phosphoribosyltransferase, and stir at 35° C. for 6 hours.

[0084] S3: After the enzymatic reaction, the pH of the solution...

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Abstract

The invention discloses an enzymatic synthesis method of nicotinamide mononucleotide. The enzymatic synthesis method comprises the following steps: S1, taking ribose-5-phosphate, cytidine monophosphate, polyphosphate and nicotinamide as raw materials, and generating nicotinamide mononucleotide under the catalytic action of ribose phosphate pyrophosphorylase, polyphosphate kinase and nicotinamide ribose phosphate transferase. The applicant uses polyphosphate kinase to catalyze cytidine monophosphate to cyclically obtain cytidine triphosphate by using a phosphate group provided by the polyphosphate, and the cytidine triphosphate is used as a pyrophosphoric acid donor of the ribose phosphate pyrophosphorylase to promote the enzymatic reaction of the ribose-5 phosphate and the nicotinamide, thereby avoiding the use of expensive ATP (adenosine triphosphate). Besides, the cytidine monophosphate and the cytidine triphosphate in the enzymatic reaction process provided by the invention are almost insoluble in water under an acidic condition, so that impurities in the product can be quickly, simply and conveniently removed by directly adjusting the pH value after the reaction is finished, the purification process is simplified, and the separation and purification difficulty is reduced.

Description

technical field [0001] This application relates to the field of biochemical technology, in particular to an enzymatic synthesis method of nicotinamide mononucleotide. Background technique [0002] Nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is the product of the reaction between nicotinamide ribokinase and nicotinamide ribose, and it is also one of the key precursors of nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NAD+). NMN exerts its physiological functions by converting into NAD+ in the human body, such as activating NAD+ substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulating cell survival and death, maintaining redox state, etc. However, the molecular weight of NAD+ is too large to be taken into cells orally, and it is difficult to meet the needs of specific target groups only relying on the micro-synthesis of cells in the body. However, with further research on NMN, it has been found that ea...

Claims

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Application Information

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IPC IPC(8): C12P19/30
CPCC12P19/305Y02P20/55
Inventor 李钊
Owner 深圳希吉亚生物技术有限公司
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