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Enzymatic synthesis method of beta-nicotinamide mononucleotide

A single nucleotide and purine nucleoside phosphorylase technology, applied in the biological field, can solve the problems of high production cost, large consumption of ATP, unstable intermediate PRPP compound and other problems in the biocatalysis method, so as to improve the substrate conversion rate, cost reduction effect

Active Publication Date: 2021-05-14
珠海瑞德林生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because nicotinamide phosphoribosyltransferase catalyzes a reversible reaction, it can also hydrolyze NMN while synthesizing NMN, and the reaction conversion rate is low
At the same time, the intermediate PRPP compound is unstable, which is not conducive to the reaction
Again, the multi-step reactions of the above two methods all require ATP to participate in the reaction, and the entire process route needs to consume a large amount of ATP, resulting in a relatively high production cost of the biocatalytic method

Method used

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  • Enzymatic synthesis method of beta-nicotinamide mononucleotide
  • Enzymatic synthesis method of beta-nicotinamide mononucleotide
  • Enzymatic synthesis method of beta-nicotinamide mononucleotide

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Experimental program
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Effect test

Embodiment 1

[0039] Source of PNP enzyme Bos taurus (Bovine) ;

[0040] The preparation source of NRK enzyme is Haemophilus influenzae (ATCC 51907);

[0041] PNP / NRK enzyme ratio 1:10, 38°C, 50mM Tris (pH8.0), pH7.0 The substrate concentration of adenosine is 250mM, the substrate concentration of nicotinamide is 250mM, 250mM sodium polyphosphate, the bottom of ATP The concentration of the substrate is 10mM, the concentration of magnesium ions is 50mM, and the conversion rate of the substrate is 90%;

[0042] Preparation of PNP enzyme: The protein sequence of PNP enzyme derived from Bos taurus is shown in Table 1. The corresponding DNA gene sequence was codon-optimized and synthesized in vitro by Anhui General Biotechnology, and digested with Nde I / Xho I (NEB Company ), and connected to the same pET28a plasmid (purchased from Addgene). Transform the constructed plasmid into the E.coli BL (DE3) strain (Shanghai Weidi Biology), and confirm that the correct colony is inoculated into the LB ...

Embodiment 2

[0048] PNPase 1 source Escherichia coli (strain K12) ;

[0049] PNP Enzyme 2 source Bacillus clausii (strain KSM-K16);

[0050]NRK enzyme source Salmonella typhimurium (ATCC 700720);

[0051] The ratio of PNP / NRK enzyme is 100:1, 35°C, 20mM PBS pH8.5, the substrate concentration of adenosine is 135mM, the substrate concentration of nicotinamide is 135mM, the substrate concentration of ATP is 20mM, and the concentration of polyphosphate is 135mM, the concentration of magnesium ions is 200mM, and the substrate conversion rate is 95%;

[0052] Preparation of PNPase 1: The protein sequence of PNPase derived from Escherichia coli (strain K12) is shown in Attached Table 1. The corresponding DNA gene sequence was codon-optimized and synthesized in vitro by Anhui General Biotechnology, and then genetic recombination was carried out according to the method of Example 1. Bacteria construction and cell culture, and the wet cells were collected by centrifugation.

[0053] Preparati...

Embodiment 3

[0058] PNP enzyme source Bacteroides fragilis (strain ATCC 25285 );

[0059] Source of NRK enzyme B. pseudomallei K96243 ;

[0060] immobilized enzyme form;

[0061] The ratio of PNP / NRK enzyme is 10:1, 40°C, immobilized enzyme, the substrate concentration of adenosine is 50mM, the substrate concentration of nicotinamide is 50mM, the substrate concentration of ATP is 5mM, the concentration of manganese ion is 5mM, six Sodium metaphosphate 80mM, PBS concentration 200mMpH7.0, substrate conversion rate 90%;

[0062] Preparation of PNP enzyme: The protein sequence of PNP enzyme derived from Bacteroides fragilis (strain ATCC 25285 ) is shown in Attached Table 1. The corresponding DNA gene sequence was codon-optimized and synthesized in vitro by Anhui General Biotechnology, and then genetic recombination was carried out according to the method of Example 1 Bacteria construction and cell culture, and the wet cells were collected by centrifugation.

[0063] Preparation of NRK enz...

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Abstract

The invention relates to the technical field of biology, and discloses an enzymatic synthesis method of beta-nicotinamide mononucleotide. According to the method, adenosine and phosphate serve as starting materials, D-ribose-1-phosphoric acid and nicotinamide ribose intermediates are generated under enzyme catalysis of an enzyme composition of PNP enzyme and NRK enzyme, NMN is obtained through an irreversible reaction finally, and the substrate conversion rate can be greatly increased; the whole reaction system only needs two enzymes to participate, and the by-product adenine can be recycled; meanwhile, only one molecule of ATP needs to be consumed while one molecule of NMN is generated, and ADP is regenerated into ATP by adding polyphosphate kinase, so that the cost of the whole process is greatly reduced.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an enzyme-catalyzed synthesis method of β-nicotinamide mononucleotide. Background technique [0002] β-nicotinamide mononucleotide (Nicotinamide mononucleotide, abbreviated as NMN) is a substance that exists in living organisms. After being adenylated by nicotinamide nucleotide adenosyltransferase, it is transformed into a biological cell for survival. An important substance of nicotinamide adenine dinucleotide (NAD+, also known as coenzyme I). In March 2017, a study by David Scinclair's research team published in "Science" showed that the increase of NAD+ in mice reversed the signs of tissue and muscle aging in older mice, which indicated that human rejuvenation is no longer a dream. Because the molecular weight of NAD+ is too large, it cannot be taken into the cells orally, and it mainly depends on the synthesis of cells in the body, and the synthesis amount is very low. However,...

Claims

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Application Information

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IPC IPC(8): C12P19/30
CPCC12P19/30
Inventor 于铁妹林立峰凌瑞枚秦国富何秀秀谭文静潘俊锋刘建
Owner 珠海瑞德林生物有限公司
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