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Innovative method for improving enzyme activity of NMN biosynthetase Nampt

A construction method and transferase technology, applied in the field of improving the enzymatic activity of NMN biosynthetic enzyme Nampt, can solve the problems of difficult industrial large-scale production, high cost of enzymatic reaction, unstable production process, etc.

Active Publication Date: 2021-07-06
HOBOOMLIFE BIO TECH SHENZHEN CO LTD
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, enzymatic reactions are mostly used to realize the synthesis of nicotinamide mononucleotide, and the natural nicotinamide phosphoribosyltransferase (NiaciNamide phosphoribosyltransferase, Nampt) has the problem of low enzymatic activity, which leads to high cost of traditional enzymatic reactions and poor reaction conditions. Harsh, unstable production process, big difference in each batch of product indicators, low reaction capacity, it is difficult to achieve large-scale factory production, which limits the large-scale application of NMN

Method used

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  • Innovative method for improving enzyme activity of NMN biosynthetase Nampt
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  • Innovative method for improving enzyme activity of NMN biosynthetase Nampt

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Experimental program
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Effect test

Embodiment 1

[0061] (1) Pipette 200 μL of glycerol-preserved strain E.coliDH5α-ppsumo-Nampt and inoculate it in 20 mL of LB medium (containing 50 μg / mL of kanamycin), and culture overnight on a constant temperature shaker at 37°C and 200 rpm. ; Utilize the Plasimid Mini Kit I (100) kit (purchased from OMEGA) to extract the plasmid;

[0062] (2) Prepare the PCR amplification reaction system (50 μL) on ice, and finally add KOD-Plus-Neo enzyme to ensure the activity of the enzyme: 1.5 μL each of mutant primers F / R (10 μM), 10×PCR Buffer for KOD- Plus-Neo 5μL, 2mM dNTPs 5μL, 25mM MgSO 4 3μL, DNA template 2 Make up 50 μL of O;

[0063] The mutation primers involved are the primers shown in Table 2, and were synthesized by Aiji Biotechnology Co., Ltd.;

[0064] PCR amplification program: pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 s, annealing at 55-65°C for 30 s, extension at 68°C for 4 min, 30 cycles; extension at 68°C for 4 min; storage at 4°C.

[0065] (3) Take 5 μL of...

Embodiment 2

[0074] Carry out SDS-PAGE electrophoresis expression verification to 10 mutants obtained in embodiment 1

[0075] 1. Induced expression

[0076] (1) Seed culture: according to the inoculum size of 1%, take 100 μ L of bacterial liquid from the glycerol tube and inoculate it into the Erlenmeyer flask (10 mL / 50 mL) of the LB liquid medium containing Kana (50 μ g / mL). 37°C, 200rmp constant temperature shaking incubator for 12h.

[0077] (2) Fermentation culture: Inoculate the seed liquid into an Erlenmeyer flask (100mL / 500mL) containing Kana-resistant LB liquid medium according to the inoculation amount of 2%, and cultivate it in a constant temperature shaker at 37°C and 180rmp for 2-3h To optical density value OD 600 When it is 0.5-0.6, add 0.25mM IPTG, and induce expression at 30°C and 180rmp for 12h.

[0078] 2. Sample pretreatment

[0079] (1) Take 1 mL of bacterial liquid in a 1.5 mL EP tube, centrifuge at 6000 rpm at 25 °C for 3 min, and discard the supernatant.

[0080...

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Abstract

The invention provides an innovative method for improving the enzyme activity of NMN (Nicotinamide Mononucleotide) biosynthetase Nampt (NiaciNamide phosphoribosyltransferase), and relates to the technical field of gene engineering. According to a mutant protein, the structure of the target protein Nampt is analyzed by utilizing FoldX and DeepDDG software, a plurality of key sites influencing enzyme functions are predicted, and enzyme semi-rational design is carried out. In the embodiment of the invention, according to a point mutation principle, primers are designed to construct 10 mutant strains, the activity of 8 mutants is higher than that of a wild type strain, the NMN yield of Nampt-V365L mutant is improved by 62%, and the NMN yields of NamptS248A, NamptN164L, NamptS382M, NamptA245T and NamptA208G are respectively improved by 34%, 27%, 27%, 22% and 17%.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an innovative method for improving the enzyme activity of NMN biosynthetic enzyme Nampt. Background technique [0002] Nicotinamide Mononucleotide (NMN) is an organic molecule and a nucleotide that can reverse aging and prolong life. [0003] At present, enzymatic reactions are mostly used to realize the synthesis of nicotinamide mononucleotide, and the natural nicotinamide phosphoribosyltransferase (NiaciNamide phosphoribosyltransferase, Nampt) has the problem of low enzymatic activity, which leads to high cost of traditional enzymatic reactions and poor reaction conditions. Harsh, unstable production process, big difference in each batch of product indicators, low reaction capacity, it is difficult to achieve large-scale factory production, which limits the large-scale application of NMN. Contents of the invention [0004] In view of this, the object ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12R1/19
CPCC12N9/1077C12N15/70C12Y204/02012C12N15/52
Inventor 赵丽青陈建生段志刚张海潮黄贝佳
Owner HOBOOMLIFE BIO TECH SHENZHEN CO LTD
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