Nicotinamide mononucleotide adenylyl transferase gene and application thereof

A technology of mononucleotide adenylyltransferase and nicotinamide adenine, which is applied in the field of genetic engineering, can solve problems that no one has studied, and achieve the effect of reducing the difficulty of the process and reducing the production cost

Inactive Publication Date: 2016-07-13
SOUTH CHINA NORMAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] In 2004, the Palmarisa research group improved the method of Tanimori et al. They used a silylating reagent to silanize nicotinamide to protect the amide bond. After the reaction, the excess silylating reagent was evaporated, and then combined with tetraacetyl ribose The reaction was carried out under the catalysis of TMSOTf, and the yield of nicotinamide ribose was 45% after deprotection of methanolamine, and the β-type compound was directly generated, which avoided the separation of the two configurations. After the amide silylation, the excess silylating reagent needs to be distilled off first, and the silylation reaction operation requires absolute anhydrous
[0020] However, no one has studied the nicotinamide mononucleotide adenylyltransferase derived from Methanothermobactersp.CaT2 and used it as an enzyme catalyst to synthesize nicotinamide adenine dinucleotide

Method used

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  • Nicotinamide mononucleotide adenylyl transferase gene and application thereof
  • Nicotinamide mononucleotide adenylyl transferase gene and application thereof
  • Nicotinamide mononucleotide adenylyl transferase gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0061] Cloning of embodiment 1 nicotinamide mononucleotide adenylyltransferase gene

[0062] The nicotinamide mononucleotide adenylyltransferase gene derived from Methanothermobactersp.CaT2 used in the present invention was synthesized by GenScript Biotechnology Co., Ltd., its nucleotide sequence is shown in SEQ ID NO: 1, and its amino acid sequence is shown in SEQ ID NO : As shown in 2, culture medium LB (g·L -1 ): Yeast extract 5g, peptone 10g, NaCl 10g, add distilled water to 1L.

[0063] The primers used to construct the expression vectors are provided with enzyme cutting sites, and the primer sequences are as follows:

[0064] The upstream primer (NMNAT-sense contains NcoI) is:

[0065] 5'-CATG CCATGG GCATGCGTGGTTTCATC-3'

[0066] Downstream primers (NMNAT-anti containing BamHI) are:

[0067] 5'-CGC GGATCC TTATTTATCGGTTTGCGCC-3'

[0068] All primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. PCR conditions of the gene: denaturation at 94°C for...

Embodiment 2

[0069] Example 2 Construction of recombinant expression vector pET-28a-NMNAT

[0070] Use NcoI and BamHI to respectively digest pET-28a (purchased from Novagen Merck China) and the amplified target gene containing two restriction sites (obtained by PCR amplification in Example 1), and recover the double-digested target gene by gel respectively. Fragment and expression vector, the expression vector pET-28a that has been double digested and target gene (gene shown in SEQIDNO: 1) carry out overnight connection with T4-DNA ligase (purchased from TaKaRa Company), obtain recombinant vector pET-28a -NMNAT; add 10 μL of the ligation product to 100 μL of Escherichia coli BL21 competent cells, place on ice for 30 min, and heat shock at 42°C for 90 s. Place on ice for 2 minutes. Add preheated 0.45mL SOC medium (2% (W / V) peptone, 0.5% (W / V) yeast extract powder, 0.05% (W / V) NaCl, 2.5mMKCl, 10mMMgCl 2 , 20 mM glucose. ). 220rpm, 37°C, 1h. Add 200 μL of the bacterial solution to an LB ...

Embodiment 3

[0071] Example 3 Induced expression of nicotinamide mononucleotide adenylyltransferase gene in Escherichia coli BL21

[0072] Pick the recombinant bacteria E.coliBL21 (containing pET-28a-NMNAT) and the control bacteria E.coliBL21 (containing pET-28a) into LB liquid medium containing 30 μg / mL kanamycin, and culture them overnight at 37°C with shaking. Then they were inoculated into fresh LB liquid medium containing 30 μg / mL kanamycin according to 2% inoculation amount, and cultured at 37°C until OD 600 At about 0.6, add IPTG to a final concentration of 0.2mmol·L -1 , 16°C, 220rpm, after induction of expression for 24h, centrifuge (4°C, 5000rpm, 15min), resuspend the bacteria sludge with 100mM Tris-HCl buffer (pH9.0), and sonicate the cells (power 300W, ultrasonic 3s, intermittent 5s, total 5min), centrifuge (4°C, 12000rpm, 15min).

[0073] Determination of supernatant enzyme activity: the enzyme reaction system includes 100mM HEPES buffer (pH7.4), 13mMMgCl 2 , 0.2mMATP, 0.2m...

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Abstract

The invention discloses a nicotinamide mononucleotide adenylyl transferase gene. The nucleotide sequence of the gene is shown as SEQ ID NO: 1. The amino acid sequence of the gene is shown as SEQ ID NO:2. The invention also discloses an expression vector; a method comprises the steps of using the expression vector for transforming the host cell, culturing a transformant, and acquiring nicotinamide mononucleotide adenylyl transferase gene from culture, so as to obtain the recombinant nicotinamide mononucleotide adenylyl transferase gene. The invention discloses application of the recombinant nicotinamide mononucleotide adenylyl transferase gene to in vitro conversion of nicotinamide mononucleoside NMN into nicotinamide adenine dinucleotide NAD<+>. The invention also discloses a synthesis method of nicotinamide adenine dinucleotide NAD<+>. The method uses nicotinamide mononucleoside NMN as raw material to generate nicotinamide adenine dinucleotide NAD<+> under the action of the recombinant nicotinamide mononucleotide adenylyl transferase gene.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a nicotinamide mononucleotide adenylyltransferase gene and its application. Background technique [0002] Nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide, NAD + ) is an indispensable small molecule compound in the living body. NAD + Dependent biomacromolecules play a very important role in life activities, participating in processes such as energy transfer, substance metabolism and signal transduction. NAD + / NADH participates in the oxidation-reduction reaction in organisms and maintains the redox balance in cells. In addition, it is a coenzyme for more than 200 reduction reactions in the living body, and it is also a type 3 NAD + A substrate for consuming enzymes that play a vital role in various cellular physiological processes, NAD + The bioenergetic state of the cell even determines the life and death of the cell. NAD + The metabolism...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/70C12P19/32C12R1/19
CPCC12N9/1241C12N15/70C12N2800/101C12P19/32C12Y207/07001
Inventor 马骥韩诗蕾王三永李春荣杨定乔
Owner SOUTH CHINA NORMAL UNIVERSITY
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