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Nicotinamide phosphoribosyltransferase mutant, recombinant expression vector and recombinant bacterium containing mutant, and application

A technology of ribose phosphoric acid and recombinant carrier, applied in the field of biotechnology, can solve the problems of low activity, large amount of organic solvent, difficult separation and purification, etc., and achieve the effects of improving enzyme activity, promoting synthesis and improving enzyme activity

Active Publication Date: 2020-09-29
仙居两山生物科技有限公司
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  • Abstract
  • Description
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AI Technical Summary

Problems solved by technology

[0004] The traditional production of NMN is obtained by chemical synthesis, using nicotinamide ribose as raw material, and phosphorylation with phosphorus oxychloride. However, the phosphorylation specificity of chemical synthesis is not high, resulting in too many impurities in the product, and separation and purification are extremely difficult. The yield is very low; at the same time, the amount of organic solvent used is large, and the environmental pollution is serious. For example, in 2002, people such as Tanimori used acetyl-protected ribose and nicotinamide to undergo a condensation reaction under the catalysis of TMSOTf; The silylating reagent silylates nicotinamide. Therefore, at present, NMN needs to be prepared by enzymatic method, in which nicotinamide phosphoribosyltransferase is the rate-limiting enzyme of the whole reaction, but the activity of the existing nicotinamide phosphoribosyltransferase is too low , making the production efficiency of NMN unsatisfactory

Method used

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  • Nicotinamide phosphoribosyltransferase mutant, recombinant expression vector and recombinant bacterium containing mutant, and application
  • Nicotinamide phosphoribosyltransferase mutant, recombinant expression vector and recombinant bacterium containing mutant, and application
  • Nicotinamide phosphoribosyltransferase mutant, recombinant expression vector and recombinant bacterium containing mutant, and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] The construction of embodiment 1 wild-type Nampt expression vector

[0072] According to the coding sequence of Luteibactersp.UNCMF366Tsu5.1 nicotinamide phosphoribosyltransferase in the GenBank database (GenBank: SFW68293, SEQ ID NO: 36), Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to synthesize the gene fragment and named it Nampt gene. In order to facilitate subsequent cloning, two bases of CC were added to the 5' end of the gene fragment to form a NcoI restriction site, and six bases of GGATCC were added to the 3' end to form a BamHI site.

[0073] The synthesized Nampt gene and pET30a vector were double-digested with NcoI and BamHI respectively, and the fragments were recovered with DNA gel kits, ligated with T4 DNA ligase, transformed into Escherichia coli DH5a, and screened with kanamycin-resistant LB plates , to obtain clones.

[0074] Eight clones were picked and the plasmids were extracted with a plasmid extraction kit, sequenced by Suzhou Jinwe...

Embodiment 2

[0075] The expression of embodiment 2 Nampt and the preparation of enzyme

[0076] The expression vector pET30a-Nampt obtained in Example 1 was transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain capable of highly expressing nicotinamide phosphoribosyltransferase.

[0077] The recombinant strain highly expressing nicotinamide phosphoribosyltransferase was fermented and cultured in a shake flask according to a conventional method. When the OD600 of the fermentation broth reached 0.7, it was induced by adding isopropylthiogalactopyranoside IPTG with a final concentration of 1 mM, and the temperature was lowered to 25 Continue culturing at ℃ for 16 hours, centrifuge at 4000 rpm for 20 minutes, collect the cells, wash twice with 50 mM PBS buffer solution with a pH of 7.4, and obtain cells containing nicotinamide phosphoribosyltransferase.

Embodiment 3

[0078] The determination of embodiment 3 nicotinamide phosphoribosyltransferase activity

[0079] The bacterium containing nicotinamide phosphoribosyltransferase collected in Example 2 was weighed 10 g, and cetyltrimethylammonium bromide CTAB with a final concentration of 0.5% was added to permeabilize the cells, and 100 mM pH of 8.0 was added. PBS buffer to a final volume of 50 mL to obtain a crude nicotinamide phosphoribosyltransferase enzyme solution.

[0080] In the 1mL reaction system, add 5-phosphoribose-1-pyrophosphate PRPP to a final concentration of 5mM, add ATP to a final concentration of 1mM, add magnesium ions to a final concentration of 16mM, add 50μL of crude enzyme solution, and add 100mM PBS buffer with a pH of 8.0 solution to a final volume of 1 mL to obtain a reaction solution.

[0081] The reaction solution was reacted at 37° C. for 30 minutes, 250 μL of the reaction solution was taken out, and 250 μL of 100 mM PBS buffer solution with a pH of 8.0 and 500 μ...

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Abstract

The invention relates to the technical field of biological science and technology and provides a nicotinamide phosphoribosyltransferase mutant, a recombinant expression vector and a recombinant bacterium containing the mutant, and application in beta-nicotinamide mononucleotide synthesis. In the nicotinamide phosphoribosyltransferase mutant, 1 or 2 amino acid residues in amino acids selected fromamino acid equivalent positions of sites 156, 174, 203, 231, 242, 258, 272 and 405 in an amino acid sequence of a wild-type nicotinamide phosphoribosyltransferase are replaced by other amino acid residues. The nicotinamide phosphoribosyltransferase mutant disclosed by the invention increases the enzyme activity of the nicotinamide phosphoribosyltransferase, can promote synthesis of a beta-nicotinamide mononucleotide and can promote production of the beta-nicotinamide mononucleotide.

Description

technical field [0001] The invention relates to the technical field of biotechnology, in particular to a mutant of nicotinamide phosphoribosyltransferase, a recombinant expression vector containing the mutant, recombinant bacteria and applications. Background technique [0002] NMN is a substance inherent in the human body and is also rich in some fruits and vegetables. In the human body, NMN is the precursor of NAD+, and its function is reflected by NAD+. NAD+ is also called coenzyme, and its full name is nicotinamide adenine dinucleotide. It exists in every cell and participates in thousands of reactions. NMN has important physiological functions for human cells. It can be synthesized naturally in cells and can also be derived from a variety of foods. , including broccoli, cabbage, cucumber, edamame, avocado, and more. In the human body, NMN is the precursor for the synthesis of NAD+, and its physiological function is mainly reflected by increasing the level of NAD+. NA...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P19/30C12R1/19
CPCC12N9/1077C12Y204/02012C12N15/70C12P19/30
Inventor 朱廷恒周敏向仲勋徐海斌
Owner 仙居两山生物科技有限公司
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