Nicotinamide phosphoribosyltransferase, encoding gene, recombinant vector and application for preparing nmn

Abstract

The invention discloses a nicotinamide phosphoribosyltransferase, coding gene, recombinant vector and application for preparing NMN, and belongs to the field of biotechnology. The enzyme is an amino acid sequence such as pSEQ ID No.1 or pSEQ ID No.2 or For the protein shown in pSEQ ID No.3 or pSEQ ID No.4, the gene encoding the above protein is cloned into an E. coli expression vector to construct a recombinant plasmid; then the recombinant plasmid is transformed into E. coli competent cells, and positive clones are selected. Obtain recombinant Escherichia coli expression strains, and then carry out liquid fermentation; during the fermentation process, the recombinant nicotinamide phosphoribosyltransferase produced can produce β-nicotinamide mononucleotide after adding substrates; the preparation of NMN by the present invention is convenient and efficient, and the fermentation The process is simple, time-consuming and low-cost. The cost of obtaining NMN is 0.25-1.0 yuan / mg. Compared with the traditional method, the cost can be reduced by more than 90%, and the output is as high as 51.75mg per g protein, with remarkable economic benefits.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to nicotinamide phosphoribosyltransferase, encoding gene, recombinant vector and application for preparing NMN. Background technique [0002] β-nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is the reaction product of nicotinamide phosphoribosyl transferase (Nicotinamide phosphate ribose transferase, Nampt) and nicotinamide, etc. (Nicotinamide adenine dinucleotide, NAD+) is a key precursor of the salvage synthesis pathway. [0003] In mammals, NMN is generated from Nicotinamide (Nam) under the catalysis of Nampt, and then NMN is catalyzed by Nicotinamide mononucleotide adenosinetransferase (Nmnat) to generate NAD+. That is, 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....

AI Technical Summary

Problems solved by technology

[0005] However, the in vitro preparation method of NMN is mainly based on chemical synthesis. For example, in 2002, Tanimori et al. used acetyl-protected ribose and nicotinamide to undergo a condensation reaction under the catalysis of TMSOTf; The reagents silanize nicotinamide, and then react with acetyl ribose under the catalysis of TMSOTf; these chemical synthesis methods have problems such as high cost, low yield and large pollution of chemical reagents

[0006] The most environmentally friendly and best way to prepare NMN is the biosynthesis method, that is, using Nampt to catalyze Nam to generate NMN; and generally using yeast to realize the synthesis of NAD precursor NMN; the existing natural Nampt has the problem of low enzyme activity, there is Long time-consuming, high cost, low yield, and difficulty in realizing large-scale factory production limit the large-scale application of NMN

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