Method for preparing nicotinamide mononucleotide

A single nucleotide and nicotinamide technology, applied in the direction of fermentation, etc., can solve the problems of high cost of NMN process, achieve the effect of reducing production cost, complete conversion of raw materials, and simple operation of synthesis reaction

Active Publication Date: 2021-01-01
湖州颐盛生物科技有限公司
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Problems solved by technology

[0006] In order to overcome the high cost of the enzymatic preparation of NMN in the prior art, the inventors found another way, and after rep

Method used

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  • Method for preparing nicotinamide mononucleotide

Examples

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Example Embodiment

[0051]Example 1 Genetic engineering construction of enzyme-producing strains

[0052]1. Nucleosidase (NCBI accession number WP_012699601.1), ribose phosphopyrophosphate kinase (amino acid sequence NCBI accession number A3MV85, BAA 05286 mutant N120S, BAA 05286 mutant L135I), nicotinamide phosphoribosyl transferase (amino acid sequence NCBI Accession numbers AAR87771, NP_717588, WP_115638626), inorganic pyrophosphatase (NCBI accession number Q58025) according to its amino acid sequence, commissioned Nanjing GenScript Biotechnology Company to optimize the nucleotide sequence to facilitate expression in E. coli. The coding genes of the above four enzymes are SEQ ID NO: 1, SEQ ID NOs: 2-4, SEQ ID NOs: 5-7 and SEQ ID NO: 9, respectively cloned into pET24a(+) NdeI and BamI restriction enzymes Cut the site. According to the conventional operation method, the 8 pET24a plasmids were transferred into BL21(DE3) competent cells by the calcium chloride method, and the plates containing kanamycin we...

Example Embodiment

[0058]Example 2 Obtaining enzyme

[0059]Inoculate the strains of each enzyme obtained in Example 1 in a test tube containing 4 mL of LB medium (yeast extract 5g / L, tryptone 10g / L, sodium chloride 10g / L.), cultivate overnight at 37°C, and cultivate Inoculate a triangle containing 1L of TB medium (yeast extract 24g / L, tryptone 12g / L, dipotassium hydrogen phosphate trihydrate 16.4 g / L, potassium dihydrogen phosphate 2.3 g / L, and glycerol 5 g / L.) Bottle, shake culture, add final concentration of 0.3mM isopropyl-β-D-thiogalactoside (IPTG) for induction, and cool to 25°C to continue culturing overnight. The precipitate is collected by centrifugation to obtain the bacterial cells containing each enzyme for use.

Example

[0060]Example 3 Preparation of nicotinamide mononucleotide using 5-phosphoribose as raw material

[0061]Add 0.187g of 5-phosphate ribose, 0.462g of adenosine triphosphate (ATP) sodium salt, 0.103g of nicotinamide, 0.03g of magnesium chloride hexahydrate and 20mL of 50mM Tris-HCl (pH7.5) into the flask, and shake well. After dissolving, adjust pH=7.5 with sodium hydroxide solution. Then add 0.1g ribose phosphopyrophosphate kinase BL21(DE3) / 2 cells and 0.3g nicotinamide phosphoribosyltransferase BL21(DE3) / 5 cells, shake in a shaker at 25°C for 8 hours, take samples, and use HPLC The concentration of NMN in the sample is 5.86 g / L.

[0062]The experimental results verified the functions of ribose phosphopyrophosphate kinase and nicotinamide phosphoribosyltransferase constructed in the above examples. They can be used to prepare NMN according to the method of CN108949865A, but because 5-phosphoribose is expensive, in the following examples This method is no longer used.

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Abstract

The invention provides a method for preparing nicotinamide mononucleotide. The method is characterized by using hypoxanthic acid and/or guanylic acid, ATP and nicotinamide as raw materials, carrying out combined catalytic reaction by using ribosidase, ribose pyrophosphate kinase and nicotinamide ribose phosphate transferase, and synthesizing by using a one-pot method to obtain nicotinamide mononucleotide. The method opens up a new way for synthesizing nicotinamide mononucleotide by an enzyme method, and significantly reduces the production cost.

Description

Technical field[0001]The invention belongs to the technical field of biocatalysis, and relates to a method for preparing nicotinamide mononucleotide, and specifically, to a new method for enzymatically synthesizing nicotinamide mononucleotide.Background technique[0002]Nicotinamide mononucleotide (β-Nicotinamide mononucleotide, referred to as β-NMN or NMN) is an endogenous substance in the human body. It is the product of nicotinamide phosphoribosyl transferase reaction and participates in intracellular nicotinamide purine dinucleoside Acid (NAD+The synthesis of) is its important and most direct prerequisite substance. In the human body, NMN is converted into NAD+Exercising its physiological functions, such as activating NAD+The substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulates cell survival and death, and maintains redox state. But it will gradually decrease with age. Thanks to NAD+The molecular weight is too large to be absorbed by the human b...

Claims

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Application Information

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IPC IPC(8): C12P19/30
CPCC12P19/30
Inventor 陶荣盛原犇犇朱傅赟郑云沈正权孙梁栋潘震华沈青胡海亮刘萍
Owner 湖州颐盛生物科技有限公司
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