Uridine phosphatase mutant and application thereof

A technology of uridine phosphatase and mutant, applied in the fields of genetic engineering and enzyme catalysis, can solve the problem of low activity of nicotinamide substrate, and achieve the effect of improving enzyme activity

Active Publication Date: 2020-10-09
ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to overcome the problem of low activity of uridine phosphatase to nicotinamide substrate, the present invention utilizes genetic engineering technology to transform and synthesize uridine phosphatase (also known as uridine phosphorylase) derived from Escherichia coli Screening, improving its nicotinamide substrate specificity and catalytic activity, and cooperating with purine nucleoside phosphorylase and nicotinamide ribokinase to achieve a multi-enzyme system to jointly catalyze the one-pot synthesis of NMN

Method used

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  • Uridine phosphatase mutant and application thereof
  • Uridine phosphatase mutant and application thereof
  • Uridine phosphatase mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Construction of wild-type uridine phosphatase gene recombinant Escherichia coli

[0048] For the wild-type uridine phosphatase derived from Escherichia coli K-12substr.MG1655 (the published amino acid GenBank sequence number is P12758), that is, SEQ ID NO: 1, codon optimization is carried out on this basis, and the whole gene synthesis coding gene sequence SEQ ID NO: 2, and design restriction endonuclease sites Nde I and XhoI at both ends of the gene, subclone into the corresponding sites of the vector pET24a (Novagen), obtain the recombinant plasmid pET24a-EcUP, see the plasmid map figure 1 . The recombinant plasmid pET24a-EcUP was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli pET24a-EcUP / BL21(DE3) expressing wild-type uridine phosphatase SEQ ID NO:1.

Embodiment 2

[0049] Example 2 Construction of Nicotinamide Ribokinase Recombinant Escherichia coli

[0050] For the nicotinamide ribokinase coding gene derived from Saccharomyces cerevisiae S288C (the published nucleic acid GenBank sequence number is NM_001182967.1), that is, SEQ ID NO: 5, the whole gene was synthesized, and restriction endonuclease sites were designed at both ends of the gene Point Nde I and XhoI, subclone into the corresponding site of the vector pET24a (Novagen), obtain the recombinant plasmid pET24a-ScNrK, see the plasmid map figure 2 . The recombinant plasmid pET24a-ScNrK was transformed into the expression host Escherichia coli BL21(DE3) to obtain the recombinant Escherichia coli pET24a-ScNrK / BL21(DE3) expressing nicotinamide ribokinase.

Embodiment 3

[0051] Example 3 Construction of uridine phosphatase mutant recombinant Escherichia coli

[0052] The wild-type uridine phosphatase was mutated by site-directed combinatorial mutation.

[0053] The pET24a-EcUP plasmid was used as a template, and 162F / 195R and 195F / 220&221R were used as primer pairs for PCR amplification. The primers were designed as follows:

[0054] 162F: 5'-GTGTTACCGCTTCTTCTGACACCGGTTACCCGGGTCAGGAAC-3',

[0055] 195F: 5'-GCAGGCTATGGGTGTTATGAACGCAGAAATGGAATCTGCTACCCTG-3',

[0056] 195R: 5'-CAGGGTAGCAGATTCCATTTCTGCGTTCATAACACCCATAGCCTGC-3',

[0057] 220&221R:

[0058] 5'-GATTTCCTGCTGGGTACGGTTAGAACCAACACCAGCAACCATACCA-3'.

[0059] The PCR reaction system includes: 50pmol each primer, 10ng plasmid template, 1×KOD neo plus buffer, 0.2mM dNTP, 1.5mM MgSO 4 , KOD neo plus 1U, add ddH 2 O to 50 μL of the total system.

[0060] The PCR amplification conditions were: 95°C for 5min; 98°C for 15s, 57°C for 30s, 68°C for 30s / kbp, 30 cycles; 68°C for 10min.

[006...

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Abstract

The invention discloses a uridine phosphatase mutant and a preparation method thereof. The amino acid sequence of the mutant is SEQ ID NO: 3, ribose-1-phosphoric acid can be effectively catalyzed to react with nicotinamide to generate nicotinamide ribose; the nicotinamide ribose can form a three-enzyme system together with purine nucleoside phosphorylase and nicotinamide ribose kinase to jointly catalyze a reaction of raw materials guanosine, phosphate, nicotinamide and ATP to synthesize the beta-nicotinamide mononucleotide by a one-pot method, and the product has good industrial development and application prospects.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and enzyme catalysis, and in particular relates to a uridine phosphatase mutant and its use in synthesizing nicotinamide ribose and beta-nicotinamide mononucleotide. Background technique [0002] β-nicotinamide mononucleotide (β-nicotinamide mononucleotide, referred to as β-NMN or NMN) widely exists in natural organisms and is the immediate precursor of NAD (nicotinamide adenine dinucleotide, also known as coenzyme I) , and NAD is an important coenzyme for many biochemical reactions in cells. After NMN is ingested into body cells, NAD can be synthesized under the catalysis of NMNAT enzyme, and its function can be reflected through NAD. According to research, NMN has the functions of enhancing immunity, anti-aging, repairing DNA damage, enhancing endurance, relieving alcohol and protecting the liver, enhancing insulin sensitivity, protecting vision and hearing, etc. [0003] Currently...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N1/21C12P19/38C12P19/30C12R1/19
CPCC12N9/1077C12P19/30C12P19/38C12Y204/02003
Inventor 范文超王金刚梁岩高书良袁圣伦任亮
Owner ZHEJIANG HUARUI BIOTECHNOLOGY CO LTD
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