Nicotinamide phosphoribosyltransferase mutant and application thereof

A ribose phosphoric acid and mutant technology, applied in the field of biological enzyme engineering, can solve the problems of low enzyme activity and inability to meet industrial production, and achieve the effects of mild reaction conditions, broad industrial application prospects, and stable energy circulation system

Active Publication Date: 2019-10-25
KINGDOMWAY BIOTECH (JIANGSU) CO LTD +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The enzymatic activity of nicotinamide phosphoribosyltransferase reported so fa

Method used

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  • Nicotinamide phosphoribosyltransferase mutant and application thereof

Examples

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Embodiment 1

[0023] Example 1 Construction of Prokaryotic Expression System

[0024] Nampt gene fragment was synthesized by Changzhou Jiyu Biotechnology Co., Ltd. and recombined into PUC57 vector. After double digestion with restriction enzymes NdeI and HindIII (purchased from New England Biolabs, NEB) at 37°C for 4 hours, 1% agarose gel electrophoresis was used for separation and gel-cutting recovery (gel recovery kit purchased from Tiangen) Biochemical Technology (Beijing) Co., Ltd.). Subsequently, it was ligated with the expression vector pET29a(+) (Novagen), which was digested with the same double restriction enzymes, at 16°C overnight under the action of T4 DNA ligase (purchased from Takara). The ligation solution was transformed into Top10 competent cells (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and colony PCR screening and sequencing were carried out to verify the positive recombinant plasmid NrK-pET29a(+).

[0025] The positive recombinant plasmid Nampt-pET...

Embodiment 2

[0027] Example 2 Preparation of enzyme freeze-dried powder by shake flask fermentation of enzyme

[0028] The above-constructed expression strains Nampt-pET29a(+) / BL21(DE3), PPK2-pET29a(+) / BL21(DE3), in 5ml LB liquid medium with a final concentration of 30μg / ml kanamycin sulfate [ 10g / l Tryptone (OXIOD), 5g / l Yeast Powder (OXIOD), 10g / l Sodium Chloride (Reagents of Chinese Medicines)] After shaking culture overnight at 37℃ and 200rpm, inoculate at 1% (V / V) ratio It was cultured in 500 ml LB liquid medium containing a final concentration of 30 μg / ml kanamycin sulfate at 37° C. and 200 rpm with shaking. When the OD600 is between 0.8-1.0, add the inducer IPTG (isopropyl-β-D-thiogalactoside, IPTG) with a final concentration of 0.1 mM, and induce overnight at 30°C. The bacteria were collected by centrifugation at 4℃ and 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 20min), centrifuged at 4℃, 12000rpm for 20min, and the supernata...

Embodiment 3

[0029] Example 3 Construction and screening of mutants

[0030] Mutant construction: use homology modeling to simulate Nampt's three-dimensional structure, and use molecular docking and the principle of lowest energy to predict the sites that may be related to catalysis and substrate binding, which are initially identified as R189, S232, R302 Three sites. Subsequently, using the Nampt-pET29a(+) recombinant plasmid as a template, NNK saturation mutations were performed on these three sites respectively (refer to Stratagene's QuikChange® Site-Directed Mutagenesis Kit operating instructions for specific mutation operations). Forward 189 mutation primer: CTGCATGATTTTGGCGCGNNKGGCGTGAGCAGCGCGGAA, reverse primer: TTCCGCGCTGCTCACGCCMNNCGCGCCAAAATCATGCAG; 232 mutation Forward primer: CGAACCGATGGCGGGCTTTNNKATTCCGGCGGCGGAACAT, reverse primer: TGTTCCGCCGCCGGAATMNNAAAGCCCGCCATCGGTTCG; 302 mutation Forward primer: GGCGCGACCGTGGTGGTGNNKCCGGATAGCGGCGATCCG, reverse primer: CGGATCGCCGCTATCCGGMNNCA...

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Abstract

The invention provides a nicotinamide ribosyltransferase mutant and application thereof. Compared with an amino acid sequence SEQID NO. 2, the difference of the amino acid sequence of the mutant is that the R189th, S232th and R302th sites in the amino acid sequence SEQID NO. 2 are subjected to single mutation or in-pair combined mutation or three combined mutation. Novel nicotinamide ribosyltransferase mutant enzyme is used for synthesis and preparation of beta-nicotinamide mononucleotide. The constructed nicotinamide ribosyltransferase mutant enzyme has the advantages that the enzyme cost islow, the transformation time is short, and the process operation is simple, and has a broad large-scale industrial application prospect.

Description

Technical field [0001] The invention relates to a novel nicotinamide phosphoribosyl transferase and its mutants, in particular to an industrial enzyme used for bioenzymatic synthesis of β-nicotinamide mononucleotide and its mutants, and belongs to the technical field of biological enzyme engineering. Background technique [0002] β-Nicotinamide mononuclotide (NMN) is an important intermediate in the synthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Studies in recent years have proved that NMN has significant anti-aging functions, so functional health foods with NMN as the active ingredient have great development potential and market prospects. At present, NMN has been approved as a health food raw material in Europe, the United States, Japan and other developed countries, and a variety of health products have been developed with NMN as the main ingredient, such as HERBALmax in the United States, GeneHarbor NMN9000 in the United States, MIRAI LAB NMN3000 capsules i...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/30C12R1/19
CPCC12N9/1077C12Y204/02012C12N15/70C12P19/30
Inventor 祝俊李斌徐飞余允东刘双喜李二军张超邢飞马晶晶张晨晨许昇
Owner KINGDOMWAY BIOTECH (JIANGSU) CO LTD
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