Method for preparing nucleosides of nicotinic acid or derivatives thereof, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide, enzyme composition and application

A technology of dinucleotide diphosphatase and nicotinic acid adenine, which is applied in the field of biomedicine, can solve problems such as environmental impact, nicotinamide riboside production, and impact, and achieve the effect of improving flexibility

Active Publication Date: 2021-07-23
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a result, the public's demand for nicotinamide riboside is gradually increasing, but the current production process is too focused on a single material as a raw material, especially when tetraacetyl ribose cannot be supplied normally, it will inevitably affect the production of nicotinamide riboside
The inability to maintain the normal supply and stable price of nicotinamide riboside will have a certain impact on the public's demand for nicotinamide adenine dinucleotide
Furthermore, the process of producing nicotinamide riboside is a chemical process, which requires the use of a large number of different organic solvents, which will have a serious impact on the environment

Method used

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  • Method for preparing nucleosides of nicotinic acid or derivatives thereof, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide, enzyme composition and application
  • Method for preparing nucleosides of nicotinic acid or derivatives thereof, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide, enzyme composition and application
  • Method for preparing nucleosides of nicotinic acid or derivatives thereof, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide, enzyme composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0216] Embodiment 1: Preparation of nicotinamide adenine dinucleotide kinase (EC 2.7.1.23)

[0217] PCR primers were designed according to the DNA sequence (SEQ ID NO.4) (gene bank accession number ARC14363.1) encoding nicotinamide adenine dinucleotide kinase in the Clostridioides difficile genome, specifically:

[0218] Upstream primer NADK1:

[0219] 5'-CTGACC GGATCC ATGAAAAGAATTATAACTATAAAT-3' (SEQ ID NO. 5)

[0220] Downstream primer NADK2:

[0221] 5'-TATGCG GAATTC CTATAAAAAATTTTTCAGATACTCT-3' (SEQ ID NO. 6)

[0222] Clostridioides difficile (Clostridioides difficile) genomic DNA was used as a template, and the above primers were used for PCR amplification to obtain the nicotinamide adenine dinucleotide kinase gene, and the PCR product was treated with restriction endonucleases BamH I and EcoRI and ligated into pET-21a to obtain pET-NADK. The recombinant expression vector is transformed into Escherichia coli HB101 to obtain a recombinant expression strain of nicoti...

Embodiment 2

[0224] Embodiment 2: Preparation of nicotinamide adenine dinucleotide diphosphatase (EC 3.6.1.22)

[0225] PCR primers were designed based on the DNA sequence (PJP11175.1) (SEQ ID NO.7) encoding nicotinamide adenine dinucleotide diphosphatase in the Saccharomyces cerevisiae genome, specifically:

[0226] Upstream primer NDP1:

[0227] 5'-CTGACC GGATCC ATGTCCACTGCAGTGACTTTTTTT-3' (SEQ ID NO. 8)

[0228] Downstream primer NDP2:

[0229] 5'-TATGCG GAATTC CTATAGATGGCTCGATGAGGTCTT-3' (SEQ ID NO. 9)

[0230] Genomic DNA of Saccharomyces cerevisiae was used as a substrate, and the above primers were used to perform PCR amplification to obtain the nicotinamide adenine dinucleotide dinucleotide diphosphatase gene, and the PCR product was treated with restriction endonucleases BamH I and EcoR I and This was ligated into pET-21a to obtain pET-NDP. The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a recombinant expression strain of nicotinamide...

Embodiment 3

[0232] Embodiment 3: Preparation of 5'-nucleotidase (EC 3.1.3.5)

[0233] PCR primers were designed based on the DNA sequence (AVB07708.1) (SEQ ID NO.10) encoding 5'-nucleotidase in the Salmonella enterica genome, specifically

[0234] Upstream primer NUCL1:

[0235] 5'-CTGACC GGATCC ATGAAAGTAAAACTGCTTGCTGCC-3' (SEQ ID NO. 11)

[0236] Downstream primer NUCL2:

[0237] 5'-TATGCG GAATTC TTACTTCTTCACATCCGCAACGCG-3' (SEQ ID NO. 12)

[0238] Using the genomic DNA of Salmonella enterica as a substrate, the 5'-nucleotidase gene was amplified by PCR with the above primers, and the PCR product was treated with restriction endonucleases BamH I and EcoR I and ligated into pET-21a to obtain pET-USHA. The recombinant expression vector was transformed into Escherichia coli HB101 to obtain a 5'-nucleotidase recombinant expression strain.

[0239] Select a single colony of the above strains and inoculate them into 4 mL of LB medium (containing 100 μg / ml ampicillin), and culture them ...

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Abstract

The invention provides a method for preparing nucleosides of nicotinic acid or derivatives thereof, nicotinic acid adenine dinucleotide and nicotinic acid mononucleotide, an enzyme composition and application. The method for preparing the nucleosides of the nicotinic acid or the derivatives thereof comprises the step of carrying out reaction by using 5'-nucleotidase and mononucleotide of the nicotinic acid or the derivatives thereof or salt of the mononucleotide as a substrate, wherein the amino acid sequence of the 5'-nucleotidase is shown as SEQ ID NO.1. Compared with a chemical synthesis method, the method is safer and more reliable, and is more environment-friendly because the use of an organic solvent can be avoided. Nicotinamide nucleoside can be more effectively produced so as to meet increasing demands.

Description

technical field [0001] The invention belongs to the field of biomedicine, and specifically relates to a method for preparing nucleosides of niacin or its derivatives, niacin adenine dinucleotide, and niacin mononucleotide, an enzyme composition and applications. Background technique [0002] With the development of science and technology in recent years, the scientific community has made major breakthroughs in pathology, genetics and other disciplines, and developed various treatment options for the "terminal diseases" that the public considered in the last century, such as cancer and AIDS, thus effectively prolonging the life expectancy of patients. life expectancy, improved quality of life and even full recovery. Therefore, the public has changed their views on such "terminal diseases" for many years, and no longer regards them as the shackles of death, rekindling the hope of life for patients and their families. At the same time, the scientific community has gained a dee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/38C12P19/30C12P19/36C12N9/12C12N9/14C12N9/16
CPCC12P19/38C12P19/30C12P19/36C12N9/16C12N9/14C12N9/1205C12Y301/03005C12Y306/01022C12Y207/01023
Inventor 潘永强卢锦春王骏
Owner BIORIGHT WORLDWIDE
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