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Nicotinamide ribose phosphate transferase mutant and application thereof

A technology of phosphoribosyl and nicotinamide, applied in the field of enzyme engineering, can solve the problems of low market competitiveness, high production cost, low enzyme activity, etc.

Active Publication Date: 2021-07-13
深圳希吉亚生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme activity of the wild-type nicotinamide phosphoribosyltransferase reported at present is generally low, resulting in high production costs and low market competitiveness, which seriously limits the industrial application of this process

Method used

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  • Nicotinamide ribose phosphate transferase mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] This embodiment provides a nicotinamide phosphoribosyltransferase, the preparation method of which is as follows:

[0056] 1. Preparation of recombinant plasmids and recombinant bacteria

[0057] The parent nicotinamide phosphoribosyltransferase used in this example is from Methanobacterium sp.PtaU1.Bin097, and its amino acid sequence is as follows (GenBank: OPY24542.1):

[0058] MNQNLLLMTDSYKASHWLQYPEGTTKIYSYIESRGGKYPETLFFGLQYLLRILEKGINEEDVWEADAFFEVHGVPFNLDGFLYIMNEHDGKLPVEIKAIAEGSVVPAHTPLLTIENTDPSCYWLTCYLETMLLRVWYPTTVATRSWYAKKIIKTYLDQTADDSEAELPSKLHDFGARGASSHESAAIGGMAHLVNFTGSDTVEGVILANKVYKCDMAAFSIPAAEHSTITAWGKENEVEAYRNMLKQFAKPNSLMAVVSDSYDIYNAVENIWGEELRQEVVDSGATIVIRPDSGHPPEIVSKVVKILDEKFGSTENSRGYRVLDNVRVIQGDGVDLDMIHEILDKLKNEGYSASNIAFGMGGYLLQKLNRDTQKFAMKCSYAKVNGKGRDVFKEPVTDKGKTSKRGRINNSLLETVFLDGEIVKEYTLDQVREKAARALE(SEQ IDNo.1)。

[0059] The nucleotide sequence encoding the nicotinamide phosphoribosyltransferase is as follows:

[0060]atgaatcaaaatttgctactgatgactgacagctataa...

Embodiment 2

[0078] Enzyme activity assay

[0079] The final concentration of preparation is 60mM nicotinamide, 25mM PRPP, 18mMMgCl 2 , 15mM KCl, 100mM Tris buffer solution, adjust the pH to 7.5. Take 4 parts of the reaction solution (900 μl each), add 100 μl of parental Nampt with the same protein concentration and supernatant crude enzyme solution of 3 kinds of mutant Nampt, react at 37 ° C for 10 min, then add 100 μl of 25% trichloroacetic acid to terminate the reaction, pass The NMN content in the reaction solution was determined by HPLC, and the specific activity of each enzyme was calculated. Taking the enzyme activity of the parental Nampt as 100% of the relative enzyme activity, the relative enzyme activity of the mutant Nampt was compared, and the results are shown in the following table:

[0080] Table 3. Relative enzyme activity detection results

[0081]

[0082] It can be seen from the above results that the enzyme activity of the single-site mutants provided in the exam...

Embodiment 3

[0084] Preparation of nicotinamide mononucleotide

[0085] Add 90mM nicotinamide, 90mM ribose-5-phosphate, 90mMATP, 20mM MgCl to the reactor 2, 100mM Tris-HCl buffer substrate solution, adjust the pH to 7.0-7.5. Then add catalytic enzyme, the addition amount is respectively: the supernatant crude enzyme liquid of the mutant V202A / L364P of 8ml / L (crude enzyme liquid / substrate solution), the phosphoric acid of 20g / L (lyophilized powder enzyme / substrate solution) Ribose pyrophosphorylase, after stirring evenly, react in a constant temperature water bath shaker. The rotating speed of the shaker was set at 50 rpm, the reaction temperature was controlled at 30°C, and the pH was maintained at 7.0-8.0. After reacting for 4 hours, a solution containing the crude product was obtained, which was filtered, purified, and dried to obtain the final product, which was confirmed to be nicotinamide mononucleotide by hydrogen spectrum and carbon spectrum examination.

[0086] In addition, the...

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Abstract

The invention discloses a nicotinamide ribose phosphate transferase mutant and application thereof. In the first aspect of the application, the nicotinamide ribose phosphate transferase mutant is provided, the nicotinamide ribose phosphate transferase mutant comprises an amino acid sequence obtained after an amino acid sequence shown as SEQ ID No.1 is mutated, and mutation comprises at least one of the facts that valine at the position 202 is mutated into alanine and leucine at the position 364 is mutated into proline. According to the embodiment of the invention, the nicotinamide ribose phosphate transferase at least has the following beneficial effects: compared with the existing nicotinamide ribose phosphate transferase, the nicotinamide ribose phosphate transferase provided by the embodiment of the invention has higher enzyme activity than the conventional wild type nicotinamide ribose phosphate transferase, and the substrate containing nicotinamide and 5-phosphoribose-1-pyrophosphoric acid can be efficiently catalytically converted into NMN.

Description

technical field [0001] The application relates to the technical field of enzyme engineering, in particular to a mutant of nicotinamide phosphoribosyltransferase and its application. Background technique [0002] Nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN) is the product of the reaction between nicotinamide phosphoribosyltransferase and nicotinamide, and it is also one of the key precursors of nicotinamide adenine dinucleotide (NAD+). NMN exerts its physiological functions by converting into NAD+ in the human body, such as activating NAD+ substrate-dependent enzyme Sirt1 (histone deacetylase, also known as sirtuin), regulating cell survival and death, maintaining redox state, etc. Because the molecular weight of NAD+ is too large, it cannot be taken into cells orally, and it mainly depends on the synthesis of cells in the body, and the synthesis amount is very low. However, with the research on the small molecule NMN, the precursor of NAD+, it has been fou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12P19/30
CPCC12N9/1077C12Y204/02012C12P19/30
Inventor 李钊
Owner 深圳希吉亚生物技术有限公司
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