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Method for preparing beta-nicotinamide mononucleotide, enzyme composition and application thereof

A mononucleotide and dinucleotide diphosphatase technology, applied in the field of biomedicine, can solve problems such as insufficient production demand, negative impact on ecology and environment, and production shortage, and achieve considerable economic results and abundant supply , cheap effect

Pending Publication Date: 2021-07-13
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The extraction method is energy-consuming and time-consuming, and has been abandoned by the industry; the chemical synthesis method needs to use a variety of organic solvents that are harmful to the environment, which is expensive and has serious negative impacts on ecology and the environment, and is also an unfeasible process.
The bio-enzyme method is cheaper and more environmentally friendly than the above two methods, but the production of various reactants such as ATP and ribose required in the enzymatic reaction has been in short supply for a long time, which is not enough to meet the production demand, and its price is therefore constantly rising. The stumbling block to the mass production of β-nicotinamide mononucleotide

Method used

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  • Method for preparing beta-nicotinamide mononucleotide, enzyme composition and application thereof
  • Method for preparing beta-nicotinamide mononucleotide, enzyme composition and application thereof
  • Method for preparing beta-nicotinamide mononucleotide, enzyme composition and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0116] Embodiment 1: Preparation of nicotinamide adenine dinucleotide diphosphatase (EC 3.6.1.22)

[0117] PCR primers were designed based on the nicotinamide adenine dinucleotide dinucleotide diphosphatase gene sequence (gene bank accession number PJP11175) in the Saccharomyces cerevisiae genome, specifically

[0118] Upstream primer NDP1:

[0119] 5'-CTGACCGGATCCATGTCCACTGCAGTGACTTTTTTT-3' (SEQ ID NO.4)

[0120] Downstream primer NDP2:

[0121] 5'-TATGCGGAATTCCTATAGATGGCTCGATGAGGTCTT-3' (SEQ ID NO. 5).

[0122] Using the genomic DNA of Saccharomyces cerevisiae as a template, PCR was performed with the above primers to amplify the nicotinamide adenine dinucleotide dinucleotide diphosphatase gene, and the PCR product was treated with restriction endonucleases BamH I and EcoR I, And connect it to pET-21a to get pET-NDP. The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of nicotinamide adenine dinucleotide...

Embodiment 2

[0124] Embodiment 2: Preparation of AMP phosphorylase (EC 2.4.2.57)

[0125] PCR primers were designed based on the AMP phosphorylase gene sequence (gene bank accession number AEH60542) in the genome of Methanosalsum zhilinae DSM 4017, specifically

[0126] Upstream primer APP1:

[0127] 5'-CTGACC GGATCC ATGCAATTAAAAGTTCAGCCAATT-3' (SEQ ID NO. 6)

[0128] Downstream primer APP2:

[0129] 5'-TATGCG GAATTC TTAAAGTTCCCTGTAAGTGGGGAC-3' (SEQ ID NO. 7).

[0130] Using the genomic DNA of Methanosalsum zhilinae DSM 4017 as a template, PCR was performed with the above primers to amplify the AMP phosphorylase gene. The PCR product was treated with restriction enzymes BamH I and EcoR I and ligated into pET-21a to obtain pET-APP. The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of AMP phosphorylase.

[0131] Select a single colony of the above strains and inoculate them into 4 mL of LB medium (containing 100 μg...

Embodiment 3

[0132] Embodiment 3: Preparation of formate dehydrogenase (EC 1.17.1.9)

[0133] PCR primers were designed based on the formate dehydrogenase gene sequence (gene bank accession number BAH57505) in the Pichia pastoris (Komagataella pastoris) genome, specifically:

[0134] Upstream primer FDH1:

[0135] 5'-CTGACCGGATCCATGAAAATCGTTCTCGTTTTGTAC-3' (SEQ ID NO.8)

[0136] Downstream primer FDH2:

[0137] 5'-TATGCGGAATTCTTATGCGACCTTTTTTGTCATTACC-3' (SEQ ID NO. 9).

[0138] Using the genomic DNA of Pichia pastoris (Komagataella pastoris) as a template, PCR was performed with the above primers to amplify the formate dehydrogenase gene, and the PCR product was treated with restriction enzymes BamH I and EcoRI and ligated into pET-21a , to obtain pET-FDH. The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of formate dehydrogenase.

[0139] Select a single colony of the above strains and inoculate them into 4 mL of LB...

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Abstract

The invention provides a method for preparing beta-nicotinamide mononucleotide, an enzyme composition and application thereof. The method comprises the following steps: 1) providing an extract of microorganisms, wherein the extract of the microorganisms contains oxidized nicotinamide adenine dinucleotide; (2) mixing nicotinamide adenine dinucleotide diphosphatase with the extract of the microorganism, and enabling the nicotinamide adenine dinucleotide diphosphatase to react with the oxidized nicotinamide adenine dinucleotide to generate beta-nicotinamide mononucleotide and AMP, wherein the amino acid sequence of the nicotinamide adenine dinucleotide diphosphatase is as shown in SEQ ID NO. 1. According to the method disclosed by the invention, the productivity can be improved, green production can be realized, and considerable economic achievements are created on the whole.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for preparing β-nicotinamide mononucleotide, an enzyme composition and applications thereof. Background technique [0002] Nicotinamide adenine dinucleotide is the most important coenzyme in living organisms. Nicotinamide adenine dinucleotide regulates energy metabolism in cells, maintains the biological clock and the normal functions of various organ cells, supports gene repair mechanisms and stabilizes telomeres, and delivers indispensable substances such as hydrogen ions to cells. Studies at home and abroad have confirmed that the level of nicotinamide adenine dinucleotide is directly related to aging and health, and the level of nicotinamide adenine dinucleotide in the body will also decrease with age, causing various Chronic disease caused by metabolic decline and genetic damage. In response to the impact of reduced levels of nicotinamide adenine dinucleotide...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/30
CPCC12P19/30
Inventor 潘永强卢锦春王骏
Owner BIORIGHT WORLDWIDE
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