CN116064469A - Nicotinamide riboside kinase mutant with improved activity and application of nicotinamide riboside kinase mutant in NMN synthesis - Google Patents

Abstract

The invention discloses a nicotinamide riboside kinase mutant with improved activity and application of the nicotinamide riboside kinase mutant to synthesis of NMN. The nicotinamide riboside kinase mutant is a mutant sequence of an amino acid sequence shown in SEQ ID NO. 2, and the mutant site at least comprises one of the following sites: bit 6, 34 or 47; alternatively, the amino acid sequence of the nicotinamide riboside kinase mutant is an amino acid sequence which at least comprises one of the mutation sites and has more than 80% identity with SEQ ID NO. 2 and has nicotinamide riboside kinase catalytic activity. By applying the technical scheme of the invention, the mutant can efficiently catalyze nicotinamide ribose and ATP to be converted into NMN, greatly reduces the cost of producing NMN by industrially applying a biocatalysis technology, and has higher industrial application value.

Description

Nicotinamide riboside kinase mutant with improved activity and application of nicotinamide riboside kinase mutant in NMN synthesis

Technical Field

The invention relates to the fields of molecular biology and bioengineering, in particular to a nicotinamide riboside kinase mutant with improved activity and application of the nicotinamide riboside kinase mutant in NMN synthesis.

Background

NAD ⁺ Is an important biological macromolecule for maintaining the vital activity of organisms, and researches show that NAD ⁺ The level is systematically reduced with age, and NAD can be recovered effectively by supplementing nicotinamide mononucleotide (nicotinamide mononucleotide, NMN) ⁺ The medicine has the effects of improving pathological states of related diseases such as metabolic diseases, aging, neurodegenerative diseases and the like, delaying aging, prolonging service life and the like. The therapeutic effects promote the development of functional foods, health products and medicaments containing NMN active ingredients, and have wide application prospects. With the increasing awareness of people on the medical and health care effects of NMN, the demand of NMN in the market is increasing.

The current method for producing NMN is mainly an enzyme catalysis method, wherein one of the methods is to specifically catalyze the conversion of nicotinamide riboside (nicotinamide riboside, NR) and ATP into NMN and ADP by nicotinamide riboside kinase (nicotinamide riboside kinase, NRK, EC 2.7.1.22). However, as the enzyme activity of the existing nicotinamide riboside kinase is low, when the nicotinamide riboside kinase is applied to industrial catalytic production of NMN, the yield is low, the production cost is high, the product lacks market competitiveness, and the industrial application of the NMN biocatalysis technology is severely restricted. Therefore, improving the catalytic activity of nicotinamide riboside kinase is a key factor for reducing the biocatalysis cost of NMN, improving the industrial application value of nicotinamide riboside kinase and promoting the application of biocatalysis technology in NMN industrial production.

Disclosure of Invention

The invention aims to provide a nicotinamide riboside kinase mutant with improved activity and application of the nicotinamide riboside kinase mutant in synthesis of NMN, so as to solve the technical problems of lower activity and lower industrial application value of nicotinamide riboside kinase in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a nicotinamide riboside kinase mutant. The nicotinamide riboside kinase mutant is a mutant sequence of an amino acid sequence shown in SEQ ID NO. 2, and the mutant site at least comprises one of the following sites: 34 th, 6 th or 47 th bit; alternatively, the amino acid sequence of the nicotinamide riboside kinase mutant is an amino acid sequence which at least comprises one of the mutation sites and has more than 80% identity with SEQ ID NO. 2 and has nicotinamide riboside kinase catalytic activity.

Further, the mutation site is I6V, S34H or T47V.

Further, the mutation sites were I6V, S H and T47V.

According to another aspect of the present invention, there is provided a DNA molecule. The DNA molecule encodes any of the nicotinamide riboside kinase mutants described above.

According to still another aspect of the present invention, there is provided a recombinant plasmid. The recombinant plasmid is connected with the DNA molecule.

Further, the vector plasmid of the recombinant plasmid is pBR327, pAT153, pUC18, pUC19, pET21 or pETite; pETite is preferred.

According to yet another aspect of the invention, a host cell is provided. The host cell contains the recombinant plasmid.

Further, host cells include prokaryotic cells or eukaryotic cells; preferably, the prokaryotic cell is BL21 (DE 3) E.coli; preferably, the eukaryotic cell is a yeast cell or a Pichia cell.

According to a further aspect of the present invention there is provided a method of producing nicotinamide mononucleotide. The method comprises the step of carrying out catalytic reaction on nicotinamide riboside and ATP or precursor substances capable of being converted into nicotinamide riboside or ATP by adopting nicotinamide riboside kinase, wherein the nicotinamide riboside kinase is any one of the nicotinamide riboside kinase mutants.

Further, the catalytic reaction includes: preparing NMN by taking nicotinamide ribose and ATP as raw materials under the catalysis of nicotinamide riboside kinase mutant; alternatively, nicotinamide, phosphoribosyl pyrophosphate and ATP are used as raw materials, and NMN is prepared under the catalysis of nicotinamide riboside kinase mutant and nicotinamide phosphoribosyl transferase; alternatively, nicotinamide, AMP and pyrophosphoric acid or salts thereof are used as raw materials, and NMN is prepared under the catalysis of nicotinamide riboside kinase mutant and adenine phosphoribosyl transferase; preferably, the nicotinamide riboside kinase mutant is used in the form of an enzyme solution, an enzyme lyophilized powder, an enzyme-containing cell, an immobilized enzyme or an immobilized enzyme-containing cell; preferably, the temperature of the catalytic reaction is 36-37 ℃; preferably, the pH of the catalytic reaction is 7.0-8.0 ℃; preferably, the concentration of nicotinamide riboside as the catalytic substrate is 5-60 mM.

By applying the technical scheme of the invention, the mutant can efficiently catalyze nicotinamide ribose and ATP to be converted into NMN, greatly reduces the cost of producing NMN by industrially applying a biocatalysis technology, and has higher industrial application value.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 shows the activity of nicotinamide riboside kinase NRK1 and its mutants.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The invention will be described in detail below with reference to the drawings in connection with embodiments.

The gene encoding human-derived nicotinamide riboside kinase NRK1 is 600 bases long (GenBank accession NO: AY611480.1, SEQ ID NO:1, ataaattattattatggaatcaattatggttgaattaaattatggcaaaattatggcaattaaattatggcaagtcaaattatgttatgttatgttatgttatgttatgttatgttatgttatgttatgttatgttatgttatgttatgatatgatatgaatgaatgaatgaatgatatgaatgatatgatatgatatgaatgatatgatatgatatgatatgaatgaatgaatgatatgaatgaatgaatgaatgaatgatatgaatgatttgatatgatatgatatgatttgatatgaatgatttgaatgaatgaatgaatgaatgaatgaatgaatgaatgatttgaatgaatgatttgaatgatttgaatgatttgatttgatttgatttgatttgatttgatttgatttgatttgatttotal-tgaatgatttgaatgatttgaatgatttgaatgatttgaatgatttgatttgaatgatttgatttgaatgatttgatttgatttgatttgatttcatgatttgatttca ctcgttatgatttgatttgatttgaatgatttgttatgttatgttatgttatgttatgttatgttatgttatgtc acid ctcgttatgttatgtotal-cgttatgtotal-cgtotal-acid-TCacid-TCtotal-TC, Q9 NW6.1, SEQ ID NO:2, MKTFIIGGVGGVGTTTLAKNLQKHLPNCSVQDDFFESEIETDKNGFLQYDEAQVLEALNMEKMMSAISSQMAARQSTDQESEIPILIEQGFNYKPLDTIWNRSYPYPYCKRRRSTRVQPPDSGYGYPQVWYPYRQRQTQTQTQTQTQTKQQVQQQVYEDLQELELAKQKCLQVTA), theoretical molecular weight of 23193.44. The crystal structure of NRK1 (PDB: 2QT 0) was resolved in 2007, asp36 and Asp56 being the active sites of the enzyme. The invention improves the catalytic activity of NRK1 by rational design and site-directed mutagenesis technology, and is used for synthesizing NMN in an industrialized and efficient way with low cost.

The inventor carries out rational design and site-directed mutagenesis after optimizing codon of original nicotinamide riboside kinase gene with nucleotide sequence shown as SEQ ID NO. 1, inserts the optimized codon into proper carrier, and screens on LB culture medium, thus obtaining nicotinamide riboside kinase mutant with high catalytic activity.

The preparation process of the nicotinamide riboside kinase mutant provided by the invention is approximately as follows: firstly rationally designing mutation sites and mutated amino acid types, optimizing codons of original nicotinamide riboside kinase genes, then artificially synthesizing full-length genes of nicotinamide riboside kinase mutants, assembling the full-length mutant genes on proper vector plasmids by a Gibson method to construct recombinant plasmids of the nicotinamide riboside kinase, converting proper host cells, and screening positive clones by Luria Broth (LB) +kanamycin culture medium. And finally, extracting plasmid DNA from positive clones, transforming expression cells, and screening by using LB+kanamycin culture medium to obtain nicotinamide riboside kinase mutants with high catalytic activity.

In a typical embodiment of the invention, a nicotinamide riboside kinase mutant is provided. The nicotinamide riboside kinase mutant is a mutant sequence of an amino acid sequence shown in SEQ ID NO. 2, and the mutant site at least comprises one of the following sites: bit 6, 34 or 47; or alternatively; the amino acid sequence of the nicotinamide riboside kinase mutant is an amino acid sequence which comprises at least one of the above mutation sites and has 80% or more (preferably 85% or more, more preferably 90% or more, most preferably 95% or more, for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, even 99.9% or more) identity with SEQ ID NO 2, i.e., a protein derived from SEQ ID NO 2 which has one or more amino acids substituted, deleted or added in the amino acid sequence defined by SEQ ID NO 2 and has nicotinamide riboside and ATP as substrates which has a higher nicotinamide riboside kinase catalytic activity than the parent of the amino acid sequence shown in SEQ ID NO 2.

By applying the technical scheme of the invention, the mutant can efficiently catalyze nicotinamide ribose and ATP to be converted into NMN, greatly reduces the cost of producing NMN by industrially applying a biocatalysis technology, and has higher industrial application value.

In a typical embodiment of the invention, the mutation site in the nicotinamide riboside kinase mutant is I6V, S34H or T47V; preferred mutation sites are I6V, S H and T47V.

According to an exemplary embodiment of the present invention, a DNA molecule is provided. The DNA molecule codes for the nicotinamide riboside kinase mutant described above. The nicotinamide riboside kinase mutant encoded by the DNA molecule has good activity.

The above-described DNA molecules of the invention may also be present in the form of "expression cassettes". "expression cassette" refers to a linear or circular nucleic acid molecule that encompasses DNA and RNA sequences capable of directing expression of a particular nucleotide sequence in an appropriate host cell. Generally, a promoter operably linked to a nucleotide of interest is included, optionally operably linked to a termination signal and/or other regulatory elements. The expression cassette may also include sequences required for proper translation of the nucleotide sequence. The coding region typically encodes a protein of interest, but also encodes a functional RNA of interest, e.g., antisense RNA or nontranslated RNA, in sense or antisense orientation. The expression cassette comprising the polynucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one other component thereof.

According to an exemplary embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the DNA molecules described above. The DNA molecule in the recombinant plasmid is placed at a proper position of the recombinant plasmid, so that the DNA molecule can be correctly and smoothly copied, transcribed or expressed.

Although the term "comprising" is used herein to define the DNA molecule, it is not intended that other sequences not functionally related thereto may be added at any one of the two ends of the DNA sequence. Those skilled in the art know that in order to meet the requirements of recombinant manipulation, it is necessary to add appropriate restriction sites for restriction enzymes at both ends of the DNA sequence, or additionally to add start codons, stop codons, etc., and thus these cases will not be truly covered if defined by a closed expression.

The term "plasmid" as used in the present invention includes any plasmid, cosmid, phage or agrobacterium binary nucleic acid molecule, preferably a recombinant expression plasmid, in double-stranded or single-stranded linear or circular form, either prokaryotic or eukaryotic, but preferably prokaryotic, in certain embodiments, any suitable vector may be used, for example: cloning vectors such as pUC18, pUC19 and the like; can be prokaryotic expression vector, pET21, pETite, etc. The invention preferably uses pETite as a vector, and host cells of the vector can be prokaryotic cells including escherichia coli or eukaryotic cells including yeast cells and Pichia pastoris cells, and the invention preferably uses HI-Control BL21 (DE 3) as an expression host cell.

According to an exemplary embodiment of the present invention, there is provided a method for producing nicotinamide mononucleotide comprising the step of catalytically reacting nicotinamide riboside with ATP using nicotinamide riboside kinase, which is any one of the nicotinamide riboside kinase mutants described above according to the present invention. The method can be a biocatalytic process or a fermentation process.

The biocatalytic process for preparing Nicotinamide Mononucleotide (NMN) specifically refers to a process for converting a biocatalytic substrate into NMN, wherein the biological enzyme is a nicotinamide riboside kinase mutant or a nicotinamide riboside kinase mutant according to the invention and one or more other enzymes are combined, and the substrate can be nicotinamide riboside and ATP, or can be a precursor substance capable of being converted into nicotinamide riboside or ATP, for example: preparing NMN by taking nicotinamide ribose and ATP as raw materials under the catalysis of the nicotinamide riboside kinase mutant; preparing NMN by taking nicotinamide, phosphoribosyl pyrophosphate (PRPP) and ATP as raw materials under the catalysis of nicotinamide riboside kinase mutant and nicotinamide phosphoribosyl transferase of the invention; NMN is prepared by taking nicotinamide, AMP and pyrophosphoric acid or salts thereof as raw materials under the catalysis of nicotinamide riboside kinase mutant and the adenosine phosphoribosyl transferase; preferably, the nicotinamide riboside kinase mutants of the invention are used in the form of an enzyme solution, an enzyme lyophilized powder, an enzyme-containing cell, an immobilized enzyme, or an immobilized enzyme-containing cell. Preferably, the temperature of the catalytic reaction is 37 ℃; the pH of the catalytic reaction is 7.0-8.0 ℃; the concentration of nicotinamide riboside as the catalytic substrate is 5-60 mM.

The advantageous effects of the present invention will be further described below with reference to examples.

The nicotinamide riboside kinase used in the following examples is obtained by artificially designing site-directed mutagenesis after optimizing codon of original nicotinamide riboside kinase with nucleotide sequence shown in SEQ ID NO. 1.

Example 1

Construction of recombinant vector and expression Strain containing parent Nicotinamide ribokinase (NRK 1) Gene

The gene sequence (GenBank accession number: AY 611480.1) of the parent nicotinamide riboside kinase (NRK 1) from the human, published in the gene library, is firstly codon optimized, the optimized gene length is 597 bases, and the sequence is SEQ ID NO 3: ATGAAGACCTTTATTATCGGTATTAGCGGTGTTACCAATAGCGGTAAAACCACACTGGCAAAAAATCTGCAGAAACATCTGCCGAATTGTAGCGTTATTAGCCAGGATGATTTTTTCAAACCGGAAAGCGAAATCGAAACCGATAAAAATGGTTTCCTGCAGTATGATGTTCTGGAAGCACTGAACATGGAAAAAATGATGAGCGCAATTAGCTGTTGGATGGAAAGCGCACGTCATAGCGTTGTTAGCACCGATCAAGAAAGCGCAGAAGAAATTCCGATTCTGATTATTGAAGGCTTCCTGCTGTTTAACTATAAACCGCTGGATACCATTTGGAACCGTAGCTATTTTCTGACCATTCCGTATGAAGAATGTAAACGTCGTCGTAGCACCCGTGTTTATCAGCCTCCGGATAGTCCGGGTTATTTTGATGGTCATGTTTGGCCGATGTATCTGAAATATCGTCAAGAAATGCAGGACATCACCTGGGAAGTTGTTTATCTGGATGGCACCAAAAGCGAAGAAGATTTATTTCTGCAGGTCTATGAGGATCTGATTCAAGAACTGGCCAAACAGAAATGTCTGCAGGTTACCGCA the full-length sequence NRK1 of NRK1 (completed by commercial Synthesis company) was then synthesized artificially. The synthesized product is recombined to a multiple cloning site of a linearization plasmid pETite C-His through a Gibson self-assembly method to obtain a recombinant plasmid pETite-nrk 1. A Turbo chemocompetent clone strain (NEB) was transformed, plated on LB agar medium containing 30. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. Plasmids were extracted from pETite-nrk/Turbo positive clone strains using the plasmid extraction kit (NEB Monorch) using the primer pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). And determining the nucleotide sequence of the parent nicotinamide riboside kinase by DNA sequencing to obtain the nucleotide sequence shown in SEQ ID NO:3, the amino acid sequence of which is shown as SEQ ID NO: 2.

Example 2

Preparation of nicotinamide riboside kinase mutant

The PCR amplification reaction system is as follows: 25. Mu.L 2X KOD One PCR Master Mix (Toyobo), 1.5. Mu.L 10. Mu. MoL/L upstream primer, 1.5. Mu.L 10. Mu. MoL/L downstream primer, 1.0. Mu.LL1.0 ng/. Mu.L pETite-nrk1 plasmid, 21. Mu.L ddH ₂ O. The PCR amplification reaction conditions were: denaturation at 98 ℃,10 seconds; annealing at 55 ℃ for 5 seconds; extension was performed at 68℃for 15 seconds, 30 cycles, and storage was performed at 4 ℃.

1. Preparation of I6V mutant

The following primer pairs were used: nrk1/I6V-F: ATGAAGACCTTTATTGTTGGTATTAGCGGTGTTACCAA (SEQ ID NO: 6), nrk1/I6V-R: CCAACAATAAAGGTCTTCATATGTATATCTCCTTCT (SEQ ID NO: 7), using the plasmid pETite-nrk1 constructed in example 1 as a template, performing high-fidelity PCR amplification of nrk/I6V mutant gene by using the PCR amplification reaction system and the PCR amplification reaction conditions, and cutting and recovering the PCR product by using a DNA gel recovery kit to obtain recombinant plasmid pETite-nrk1/I6V. The Turbo chemocompetent clone strain was then transformed, plated on LB agar medium containing 50. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. Plasmid extraction kit was used to extract plasmid of pETite-nrk/I6V/Turbo positive clone strain, using primer pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). The nucleotide sequence of the amide ribose kinase mutant I6V is determined by DNA sequencing.

2. Preparation of S34H mutant

The following primer pairs were used: nrk1/S34H-F:5'AGCGTTATTCATCAGGATGATTTTTTCAAACCGG 3' (SEQ ID NO: 8), nrk1/S34H-R:5'TCATCCTGATGAATAACGCTACAATTCGGCA 3' (SEQ ID NO: 9), using the plasmid pETite-nrk1 constructed in example 1 as a template, performing high-fidelity PCR amplification of nrk/S34H mutant genes by using the PCR amplification reaction system and the PCR amplification reaction conditions, and cutting and recovering PCR products by using a DNA gel recovery kit to obtain recombinant plasmid pETite-nrk1/S34H. The Turbo chemocompetent clone strain was then transformed, plated on LB agar medium containing 50. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. Plasmid extraction kit was used to extract plasmid of pETite-nrk1/S34H/Turbo positive clone strain, using primer pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). The nucleotide sequence of the mutant S34H is determined by DNA sequencing.

3. Preparation of T47V mutant

The following primer pairs were used: nrk1/T47V-F:5'GATAAAAATGGTTTCCTGCAGTATGATGTTC 3' (SEQ ID NO: 10), nrk1/T47V-R: the plasmid pETite-nrk1 constructed in example 1 is used as a template for carrying out high-fidelity PCR amplification of nrk/T47V mutant genes by using the PCR amplification reaction system and the PCR amplification reaction conditions, and the PCR product is cut and recovered by using a DNA gel recovery kit to obtain the recombinant plasmid pETite-nrk1/T47V. The Turbo chemocompetent clone strain was then transformed, plated on LB agar medium containing 50. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. Plasmid extraction kit was used to extract plasmid of pETite-nrk1/T47V/Turbo positive clone strain, using primer pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). The nucleotide sequence of the amide ribose kinase mutant T47V is determined by DNA sequencing.

4. Preparation of I6V+S34H+T47V mutant

The full-length sequence nrk/I6V+S33H+T47V was synthesized manually (by commercial Synthesis). The synthesized product is recombined to a multiple cloning site of a linearization plasmid pETite C-His through a Gibson self-assembly method to obtain a recombinant plasmid pETite-nrk 1/I6V+S33H+T47V. A Turbo chemocompetent clone strain (NEB) was transformed, plated on LB agar medium containing 30. Mu.g/ml kanamycin, and cultured overnight at 37 ℃. Plasmids were extracted from pETite-nrk/I6V+S33H+T47V/Turbo positive clone strain using plasmid extraction kit (NEB Monarch) using primer pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). The nucleotide sequence of the amide ribose kinase mutant I6V+S34H+T47V is determined by DNA sequencing.

Example 3

Inducible expression and isolation and purification of enzymes

Respectively transforming the plasmid pETite-nrk1 containing the parent nicotinamide riboside kinase gene and the plasmid pETite-nrk1/I6V, pETite-nrk1/S34, ETite-nrk1/T47V and nrk/I6 V+S34H+T47V containing the nicotinamide riboside kinase mutant gene into expression strainsCells E.coli HI-Control BL21 (DE 3) were incubated on LB plates containing 50. Mu.g/mL kanamycin for about 20 hours at 37 ℃. The monoclonal was inoculated into 50mL of LB liquid medium containing 50. Mu.g/mL kanamycin and cultured at 37℃for about 12 to 18 hours (OD ₆₀₀ To 0.4), and obtaining the first-level seed liquid. According to the following steps: 100 volume ratio the primary seed solution was inoculated into 100mL of LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃and 270rpm for 3 to 4 hours (OD ₆₀₀ To 0.4), and obtaining the secondary seed liquid. According to the following steps: 100 volume ratio of the secondary seed solution was inoculated into 500mL of LB liquid medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃and 200rpm for 3 to 4 hours (OD ₆₀₀ To 0.4), then cooled to 18 ℃, added with isopropyl-beta-D thiogalactoside (IPTG) inducer, and cultured for 16-18 hours at 18 ℃ and 200rpm to induce NRK1 and mutant enzyme expression thereof. Centrifugation was performed at 5000rpm for 5 minutes at 4℃to collect the cells; then, the cells were resuspended in 6 volumes of PBS buffer, washed, centrifuged at 5000rpm for 5 minutes at 4℃to collect the cells, and resuspended in 50mM Tris-HCl,100mM NaCl buffer, pH 7.4 to obtain a resuspended cell solution. Then using an ultrahigh pressure low temperature cell disruption instrument to break cells, releasing target enzyme, centrifuging at 4 ℃ and 12000rpm for 10 minutes, collecting supernatant, and obtaining crude enzyme liquid of the parent nicotinamide riboside kinase NRK1 and series mutants thereof for the next enzyme separation and purification and activity determination.

Example 4

Separation and purification of enzymes

The crude enzyme solution was filtered using a filter membrane having a pore size of 0.45. Mu.M, and was isolated and purified by a protein purification apparatus and nickel affinity chromatography. The balance A is 50mM Tris-HCl,100mM NaCl,5mM Imidiazole,pH 7.4; eluent B was 100% imidazole solution, pH 7.4. The elution peak of 30% B eluent (namely 0.3M imidazole) with the flow rate of 2.0mL/min is taken as the target protein peak, and then HiPrep 26/10 Desalong Desalting column is used for removing imidazole, so that NRK1 with higher purity and mutant enzyme thereof are obtained. Enzyme activity was determined using nicotinamide riboside and ATP as substrates. Enzyme concentration was determined by Bradford method using BSA (bovine serum albumin) as a protein standard.

Example 5

Activity measurement of enzyme

The following solutions were prepared with 50mM Tris-HCl+50mM NaCl pH 7.4: 60mM nicotinamide riboside, 60mM ATP, 100mM MgCl ₂ 60. Mu.L of each of the purified enzyme solutions obtained in example 4 was mixed and then reacted with shaking at 240rpm at 37℃for 10 minutes, followed by adding 800. Mu.L of acetonitrile water (V: V=8:2) to terminate the reaction. The content of substrate nicotinamide riboside and product nicotinamide mononucleotide was detected by high performance liquid chromatography Waters Alliance e2695/2998 and Atlantis HILIC Silica columns (Waters, 4.6X105 mm,5.0 μm particle size). The sample injection amount is 2 mu L, and the flow rate is 1.2mL min ^-1 Column temperature was 28 ℃, mobile phase a:0.1% (g/L) ammonium acetate+5% (V: V) acetonitrile water, mobile phase B: acetonitrile, gradient elution curve is 0-7min 75% B,7.1-13min 40% B,13.1-24min 75% B, flow rate is 1.2mL/min, detection wavelength is 254nm. The retention times of nicotinamide riboside, ATP, and nicotinamide mononucleotide are about 4.0min, 7.3min, and 9.0min, respectively. The contents of substrate nicotinamide riboside and product Nicotinamide Mononucleotide (NMN) in the reaction solution were detected by High Performance Liquid Chromatography (HPLC). The enzyme activity was calculated as shown in FIG. 1. Definition of one enzyme activity unit (U) of nicotinamide riboside kinase: the amount of nicotinamide riboside kinase required to convert 1.0. Mu. Mol nicotinamide riboside per minute at 37℃and pH 7.4 is one enzyme activity unit. The specific enzyme activity (U/mg) is expressed as the unit of enzyme activity per mg of protein. The enzymatic activity of the mutants was also examined in the same manner. As shown in FIG. 1, the results show that the enzyme catalytic activities of the mutants I6V, S34H, T47V, I V+S34H+T47V using nicotinamide riboside and ATP as substrates are 2.5 times, 3.6 times, 2.3 times and 5.5 times of the parent respectively, and the mutant I6V, S, H, T, V, I V+S34H+T47V can be applied to the process for preparing nicotinamide mononucleotide.

Example 6

The reaction process for synthesizing NMN comprises the following steps: take the catalytic reaction of mutant I6V+S33H247V of NRK1 as an example

In a reaction vessel, 60mL of 60mM nicotinamide riboside and 105mL of 0.1M ATP (excess), 15mL of 0.8M MgCl were added as substrate solutions ₂ (excess) then mutant I6V+S33H+T47V purified enzyme was added at a concentration of 3.0mg/mL20mL of solution, uniformly mixing, maintaining the pH value to be 7.0-7.4, controlling the reaction temperature to be 37 ℃ and the rotating speed to be 180rpm, and feeding 0.92g of nicotinamide riboside respectively in 30min and 60 min. After 100min of reaction, according to the HPLC detection result, the conversion rate of the substrate nicotinamide riboside is 72%, NMN crude product solution (containing 54mM NMN) is obtained, and NMN can be obtained after centrifugation, purification and drying.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. The nicotinamide riboside kinase mutant with the improved activity is characterized in that the nicotinamide riboside kinase mutant is a mutant sequence of an amino acid sequence shown in SEQ ID NO. 2, and the mutant site at least comprises one of the following sites: 34 th, 6 th, or 47 th; or the amino acid sequence of the nicotinamide riboside kinase mutant is at least one of the mutation sites, has more than 80% of identity with SEQ ID NO. 2 and has the nicotinamide riboside kinase catalytic activity.

2. The nicotinamide riboside kinase mutant of claim 1, wherein the mutation site is I6V, S H or T47V.

3. The nicotinamide riboside kinase mutant of claim 2, wherein the mutation sites are I6V, S H and T47V.

4. A DNA molecule encoding a nicotinamide riboside kinase mutant according to any one of claims 1 to 3.

5. A recombinant plasmid, wherein the recombinant plasmid is linked to the DNA molecule of claim 4.

6. The recombinant plasmid according to claim 5, wherein the vector plasmid of the recombinant plasmid is pBR327, pAT153, pUC18, pUC19, pET21 or pETite; pETite is preferred.

7. A host cell comprising the recombinant plasmid of claim 5 or 6.

8. The host cell of claim 7, wherein the host cell comprises a prokaryotic cell or a eukaryotic cell; preferably, the prokaryotic cell is BL21 (DE 3) E.coli; preferably, the eukaryotic cell is a yeast cell or a pichia pastoris cell.

9. A method of producing nicotinamide mononucleotide comprising catalytically reacting nicotinamide riboside and ATP, or a precursor substance capable of being converted to nicotinamide riboside or ATP, with nicotinamide riboside kinase, characterized in that the nicotinamide riboside kinase is a nicotinamide riboside kinase mutant according to any one of claims 1 to 3.

10. The method of claim 9, wherein the catalytic reaction comprises: preparing NMN by taking nicotinamide ribose and ATP as raw materials under the catalysis of a nicotinamide riboside kinase mutant;

alternatively, nicotinamide, phosphoribosyl pyrophosphate and ATP are used as raw materials, and NMN is prepared under the catalysis of the nicotinamide riboside kinase mutant and nicotinamide phosphoribosyl transferase;

alternatively, nicotinamide, AMP and pyrophosphoric acid or salts thereof are used as raw materials, and NMN is prepared under the catalysis of the nicotinamide riboside kinase mutant and adenine phosphoribosyl transferase;

preferably, the nicotinamide riboside kinase mutant is used in the form of a purified enzyme solution, an enzyme lyophilized powder, an enzyme-containing cell, an immobilized enzyme or an immobilized enzyme-containing cell;

preferably, the temperature of the catalytic reaction is 36-37 ℃;

preferably, the pH of the catalytic reaction is 7.0-8.0 ℃;

preferably, the concentration of nicotinamide riboside as the catalytic substrate is 5-60 mM.

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