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Fusion enzyme for producing nicotinamide mononucleotide and application thereof

A single nucleotide and nicotinamide technology, applied in the biological field, can solve the problem of not being able to significantly increase the expression of nicotinamide mononucleotide, and achieve the effect of improving enzyme conversion efficiency and simplifying the process

Pending Publication Date: 2022-03-11
南宁邦尔克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Previous studies have shown that Escherichia coli containing bicistronic nicotinamide phosphoribosyltransferase and ribose phosphate pyrophosphate kinase did not significantly increase nicotinamide monocistronic activity compared with Escherichia coli containing nicotinamide phosphoribosyltransferase monocistronic The expression level of nucleotide

Method used

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  • Fusion enzyme for producing nicotinamide mononucleotide and application thereof
  • Fusion enzyme for producing nicotinamide mononucleotide and application thereof
  • Fusion enzyme for producing nicotinamide mononucleotide and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0023] In this example, a fusion enzyme expression element consisting of nicotinamide phosphoribosyltransferase gene from Haemophilus ducreyi, linking peptide and ribose phosphate pyrophosphate kinase from Bacillus amyloliquefaciens was cloned into the large intestine Bacillus expression plasmid pSE380.

[0024] Construction of recombinant plasmid pSE380-NL1P

[0025] The following DNA fragments are artificially synthesized:

[0026]

[0027]

[0028]

[0029] The synthesized DNA fragment and plasmid pSE380 were double digested with restriction endonucleases EcoR I and Pst I, then ligated with T4 ligase, transformed into Escherichia coli DH5α competent cells, and the recombinant plasmid pSE380-NL1P was obtained after screening and identification .

[0030] 2. Construction of recombinant plasmid pSE380-NL2P

[0031] The following DNA fragments are artificially synthesized:

[0032]

[0033] The synthesized DNA fragment and plasmid pSE380-NL1P were double digeste...

Embodiment 2

[0041] In this example, the fusion enzyme in Example 1 was cloned into the Bacillus subtilis integration plasmid pMLK83.

[0042] 1. Construction of recombinant plasmid pMLK83-amyM

[0043] According to the promoter amyM sequence annotated in Genbank, the upstream primer was designed as 5'cccaagcttctgtacacttgcgtcctcca 3' and the downstream primer as 5'cgggatcctctcctcccctttcaa tgtg 3'. PCR reaction system 100ul: DNA template (Bacillus licheniformis ATCC 14580 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ulPrimeSTAR HS DNA polymerization Enzyme 1ul, add ddH2O to 100ul. PCR reaction program: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, and 72°C for 30s; 72°C for 10min; store at 4°C. The PCR fragment and plasmid pMLK83 were double digested with restriction endonucleases BamHI and Hind III respectively, then ligated with T4 ligase, transformed into Escherichia coli DH5ɑ, and the recombina...

Embodiment 3

[0050] Construction of recombinant Bacillus subtilis and expression of nicotinamide mononucleotide

[0051] Using conventional transformation methods, pMLK83-amyM-NL1P was transformed into Bacillus subtilis 1A751, and the bacterial solution was coated with neomycin (20ug / ml) LB plates, and amylase-deficient transformants were screened to obtain the Bacillus subtilis genetically engineered strain 1A751 [amyM -NL1P].

[0052] 1A751[amyM-NL2P], 1A751[amyM-NL3P] and 1A751[amyM-N] can be obtained by the same method.

[0053] The above-mentioned genetically engineered strains were respectively inserted into basal salt medium (containing 10ug / ml neomycin and 0.5% nicotinamide), and after culturing for 30 hours, the content of nicotinamide mononucleotide in the cells was measured.

[0054] At the same time, E. coli DH5ɑ recombinant strains containing pSE380-N, pSE380-N1P, pSE380-N2P and pSE380-N3P were inserted into basal salt medium (containing 100 mg / ml ampicillin and 0.5% nicotina...

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Abstract

The invention relates to the technical field of biology, and discloses a fusion enzyme for producing nicotinamide mononucleotide and application thereof, the amino acid sequence of the fusion enzyme is formed by combining nicotinamide phosphoribose transferase, ribose phosphate pyrophosphate kinase and a connecting peptide, and combining the nicotinamide phosphoribosyltransferase and the ribosyl pyrophosphate kinase into a fusion enzyme through a connecting peptide. The fusion enzyme gene can be transformed into escherichia coli or bacillus subtilis, and nicotinamide mononucleotide is produced through fermentation by taking the bacterium as a strain and nicotinamide as a substrate; or after the fusion enzyme is expressed in escherichia coli or bacillus subtilis, the fusion enzyme is extracted and purified, nicotinamide mononucleotide is produced through in-vitro conversion, and the fusion enzyme can remarkably improve the production efficiency of the nicotinamide mononucleotide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion enzyme for producing nicotinamide mononucleotide and its application. Background technique [0002] Cardiovascular disease is one of the most threatening diseases to human health and life in modern society. At the same time, common diseases and symptoms of aging, such as Alzheimer's disease, osteoporosis, sarcopenia, and decreased liver function, are all associated with reduced capillaries. Studies have found that nicotinamide adenine dinucleotide (nad+, also known as coenzyme I) is a key substance, and in normal age growth in animals and humans, the bioavailability and related metabolism of nad+ in cells are Gradual decline, and then lead to physiological aging. [0003] A number of studies have begun to explore how to delay aging by increasing the activity of nad+ in the body. A nad+ precursor, nicotinamide mononucleotide (NMN), shows great promise. In animal model exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N9/12C12N15/70C12N15/75C12P19/30C12R1/19C12R1/125
CPCC12N9/1077C12N9/1235C12N15/70C12N15/75C12P19/30C12Y204/02012C12Y207/06001C07K2319/00
Inventor 李晓明韦航李丛卢运琨周志强梁树华陆迪于春娇
Owner 南宁邦尔克生物技术有限责任公司
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