Nicotinamide ribokinase mutant and application thereof

A technology of nicotinamide ribose and ribokinase, which is applied in the field of biological enzyme engineering, can solve the problems of less research and development, limited application, etc., and achieves the effects of mild reaction conditions, stable energy circulation system, and broad industrial application prospects.

Active Publication Date: 2019-10-25
JIANGSU CHENGXIN PHARMA
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AI Technical Summary

Benefits of technology

This new technology allows for faster production of nerve cells called neurons that have potential use in treating neurologically related disorders such as Alzheimer' disease or Parkinsonism. It involves converting 1-5% (vol) of an organic substance from its original form into nicotinamide adenine dinucleotide phosphoribosyl transferase (NNT), allowing it to be used more efficiently than traditional methods like chemotherapy.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the efficiency at producing pure or highly concentrating nitrogen compounds from natural sources like plants without causing unwanted side effects on their environment during manufacturing processes for these substances.

Method used

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  • Nicotinamide ribokinase mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Construction of Prokaryotic Expression System:

[0024] The NrK gene fragment was synthesized by Changzhou Jiyu Biotechnology Co., Ltd. and recombined into the 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 Nr...

Embodiment 2

[0027] Example 2 Enzyme shake flask fermentation to prepare enzyme freeze-dried powder:

[0028] The above-constructed expression strains NrK-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 the ratio of 1% (V / V) 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 cells were collected by centrifugation at 4°C and 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 20min), centrifuged at 4°C, 12000rpm for 20min, and the supernatant wa...

Embodiment 3

[0029] Example 3 Construction and screening of mutants:

[0030] Mutant construction: The method of homology modeling is used to simulate the three-dimensional structure of NrK, and the sites that may be related to catalysis and substrate binding are predicted using the principle of molecular docking and lowest energy, which are initially identified as D45, D58, R161 , Y164 four sites. Subsequently, using the NrK-pET29a(+) recombinant plasmid as a template, NNK saturation mutations were performed on these four sites respectively (refer to Stratagene's QuikChange® Site-Directed Mutagenesis Kit operating instructions for specific mutation operations). 45 mutation Forward primer: GATGATTTTTATAAACCGNNKAGCGAAATTCCGATTAACG, reverse primer: CGTTAATCGGAATTTCGCTMNNCGGTTTATAAAAATCATC; 58-point mutant forward primer: CGAAAAATATGGCGTGGCGNNKTGGGATTGCCCGGAAGCG, reverse primer: CGCTTCCGGGCAATCCCAMNNCGCCACGCCATATTTTTCG; 161-point mutant forward primer: GCCGCCGCCGCCATGCGNNKGCGGGCTATAAAACCCTGGAAG,...

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Abstract

The invention provides a nicotinamide ribokinase 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 D45th, D58th, R161th and Y164 sites in the amino acid sequence SEQID NO. 2 are subjected to single mutation or in-pair combined mutation or three combined mutation or four combined mutation. Novel nicotinamide ribokinase mutant industrial enzyme is used for synthesis and preparation of beta-nicotinamide mononucleotide. The constructed nicotinamide ribokinase mutant enzyme has the advantages that the enzyme cost is low, the transformation time is short, and the process operation is simple, and has a broad large-scale industrial application prospect.

Description

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Claims

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Application Information

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Owner JIANGSU CHENGXIN PHARMA
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