Nicotinamide riboside kinase mutant with improved activity and its application in synthesizing nmn

Abstract

The application discloses a nicotinamide riboside kinase mutant with improved activity and application thereof in synthesizing NMN. The nicotinamide riboside kinase mutant is a mutant sequence of the amino acid sequence shown in SEQ ID NO:2, and the mutation site at least includes one of the following positions: the 6th position, the 34th position or the 47th position; or the amino acid sequence of the nicotinamide riboside kinase mutant is an amino acid sequence which at least includes one of the above-mentioned mutation sites and has more than 80% identity with SEQ ID NO:2 and has the catalytic activity of nicotinamide riboside kinase. According to the technical scheme of the application, the mutant can efficiently catalyze the conversion of nicotinamide riboside and ATP into NMN, greatly reduces the cost of producing NMN by using the biological catalysis technology in industry, and has high industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and bioengineering, and in particular to an enhanced nicotinamide ribokinase mutant and its application in synthesizing NMN. Background Technology

[0002] NAD + It is an important biological macromolecule that maintains the life activities of organisms. Studies have found that NAD+ + NAD levels decline systematically with age, and supplementation with nicotinamide mononucleotide (NMN) can effectively restore NAD levels. + It can improve the pathological state of metabolic diseases, aging, and neurodegenerative diseases, and has the effects of delaying aging and prolonging life. These therapeutic effects make the development of functional foods, health products, and drugs containing NMN active ingredients a promising prospect. As people's understanding of the medicinal and health benefits of NMN continues to increase, the market demand for NMN is growing daily.

[0003] Currently, the main method for producing NMN is enzymatic catalysis, one of which involves using nicotinamide riboside kinase (NRK, EC 2.7.1.22) to specifically catalyze the conversion of nicotinamide riboside (NR) and ATP into NMN and ADP. However, due to the low enzymatic activity of existing nicotinamide riboside kinases, the yield is low when applied to industrial-scale NMN production, resulting in high production costs and a lack of market competitiveness, severely restricting the industrial application of NMN biocatalysis technology. Therefore, improving the catalytic activity of nicotinamide riboside kinase is a key factor in reducing the biocatalytic cost of NMN, increasing the industrial application value of nicotinamide riboside kinase, and promoting the application of biocatalysis technology in the industrial production of NMN. Summary of the Invention

[0004] The present invention aims to provide a mutant of nicotinamide ribokinase with enhanced activity and its application in synthesizing NMN, in order to solve the technical problems of low activity and low industrial application value of nicotinamide ribokinase in the prior art.

[0005] To achieve the above objectives, according to one aspect of the present invention, a nicotinamide ribokinase mutant is provided. The nicotinamide ribokinase mutant is a mutated sequence of the amino acid sequence shown in SEQ ID NO:2, wherein the mutation site includes at least one of the following sites: position 34, position 6, or position 47; or, the amino acid sequence of the nicotinamide ribokinase mutant is an amino acid sequence that includes at least one of the above-mentioned mutation sites, has more than 80% identity with SEQ ID NO:2, and possesses nicotinamide ribokinase catalytic activity.

[0006] Furthermore, the mutation sites are I6V, S34H, or T47V.

[0007] Furthermore, the mutation sites are I6V, S34H, and T47V.

[0008] According to another aspect of the invention, a DNA molecule is provided. This DNA molecule encodes any of the aforementioned nicotinamide ribokinase mutants.

[0009] According to another aspect of the present invention, a recombinant plasmid is provided. This recombinant plasmid is linked to the aforementioned DNA molecule.

[0010] Furthermore, the vector plasmid for the recombinant plasmid is pBR327, pAT153, pUC18, pUC19, pET21, or pETite; preferably pETite.

[0011] According to another aspect of the present invention, a host cell is provided. This host cell contains the aforementioned recombinant plasmid.

[0012] Furthermore, the host cell includes prokaryotic cells or eukaryotic cells; preferably, the prokaryotic cell is BL21(DE3) Escherichia coli; preferably, the eukaryotic cell is yeast cell or Pichia pastoris cell.

[0013] According to another aspect of the present invention, a method for producing nicotinamide mononucleotide is provided. The method includes catalyzing a reaction between nicotinamide ribokinase and ATP, or a precursor that can be converted into nicotinamide ribokinase or ATP, wherein the nicotinamide ribokinase is any of the aforementioned nicotinamide ribokinase mutants.

[0014] Further, the catalytic reaction includes: preparing NMN using nicotinamide ribose and ATP as raw materials under the catalysis of a nicotinamide ribokinase mutant; or preparing NMN using nicotinamide, phosphoribosyl pyrophosphate, and ATP as raw materials under the catalysis of a nicotinamide ribokinase mutant and nicotinamide phosphoribosyltransferase; or preparing NMN using nicotinamide, AMP, and pyrophosphate or their salts as raw materials under the catalysis of a nicotinamide ribokinase mutant and adenine phosphoribosyltransferase; preferably, the nicotinamide ribokinase mutant is used in the form of enzyme solution, enzyme lyophilized powder, enzyme-containing cells, immobilized enzyme, or immobilized enzyme-containing cells; preferably, the temperature of the catalytic reaction is 36–37°C; preferably, the pH of the catalytic reaction is 7.0–8.0°C; preferably, the concentration of the nicotinamide ribose substrate is 5–60 mM.

[0015] By applying the technical solution of this invention, the mutant can efficiently catalyze the conversion of nicotinamide ribose and ATP into NMN, which greatly reduces the cost of industrial NMN production using biocatalysis technology and has high industrial application value. Attached Figure Description

[0016] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0017] Figure 1 The activity of nicotinamide ribokinase NRK1 and its mutants was demonstrated. Detailed Implementation

[0018] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0019] The gene encoding nicotinamide ribokinase NRK1, derived from humans, is 600 bases long (GenBank accession number: AY611480.1, SEQ ID NO:1). The full-length gene encodes 199 amino acids (sequence number: Q9NWW6.1, SEQ ID NO:2, MKTFIIGISGVTNSGKTTLAKNLQKHLPNCSVISQDDFFKPESEIETDKNGFLQYDVLEALNMEKMMSAISCWMESARHSVVSTDQESAEEIPILIIEGFLLFNYKPLDTIWNRSYFLTIPYEECKRRRSTRVYQPPDSPGYFDGHVWPMYLKYRQEMQDITWEVVYLDGTKSEEDLFLQVYEDLIQELAKQKCLQVTA), with a theoretical molecular weight of 23193.44. The crystal structure of NRK1 (PDB: 2QT0) was resolved in 2007, and Asp36 and Asp56 are the active sites of the enzyme.This invention modifies and enhances the catalytic activity of NRK1 through rational design and site-directed mutagenesis technology, enabling the industrial-scale, efficient, and low-cost synthesis of NMN.

[0020] The inventors optimized the codons of the original nicotinamide ribokinase gene, as shown in SEQ ID NO:1, and then rationally designed and performed site-directed mutagenesis, inserting the mutant into an appropriate vector and screening it on LB medium to obtain a nicotinamide ribokinase mutant with high catalytic activity.

[0021] The preparation process of the nicotinamide ribokinase mutant provided by this invention is roughly as follows: First, the mutation site and the types of mutated amino acids are rationally designed, and the codons of the original nicotinamide ribokinase gene are optimized. Then, the full-length gene of the nicotinamide ribokinase mutant is artificially synthesized. The full-length mutant genome is then inserted into an appropriate vector plasmid using the Gibson method to construct a recombinant plasmid of nicotinamide ribokinase, which is then transformed into appropriate host cells. Positive clones are screened using Luria broth (LB) + kanamycin medium. Finally, plasmid DNA is extracted from the positive clones, transformed into expression cells, and screened using LB + kanamycin medium to obtain the nicotinamide ribokinase mutant with high catalytic activity.

[0022] In a typical embodiment of the present invention, a nicotinamide ribokinase mutant is provided. This nicotinamide ribokinase mutant is a mutant sequence of the amino acid sequence shown in SEQ ID NO:2, wherein the mutation site includes at least one of the following sites: position 6, position 34, or position 47; or; the amino acid sequence of the nicotinamide ribokinase mutant is an amino acid sequence that includes at least one of the above mutation sites and has at least 80% (preferably 85%, more preferably 90%, most preferably 95%, for example, it can be 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 even 99.9%) identity with SEQ ID NO:2 and possesses nicotinamide ribokinase catalytic activity, i.e., in SEQ ID NO:2. A protein derived from SEQ ID NO:2 that has one or more amino acids substituted, deleted, or added in the amino acid sequence defined by NO:2 and has higher nicotinamide ribokinase catalytic activity than the parent protein with an amino acid sequence as shown in SEQ ID NO:2, using nicotinamide ribose and ATP as substrates.

[0023] By applying the technical solution of this invention, the mutant can efficiently catalyze the conversion of nicotinamide ribose and ATP into NMN, which greatly reduces the cost of industrial NMN production using biocatalysis technology and has high industrial application value.

[0024] In a typical embodiment of the present invention, the mutation site in the nicotinamide ribokinase mutant is I6V, S34H or T47V; preferably, the mutation sites are I6V, S34H and T47V.

[0025] According to a typical embodiment of the present invention, a DNA molecule is provided. This DNA molecule encodes the aforementioned nicotinamide ribokinase mutant. The nicotinamide ribokinase mutant encoded by this DNA molecule exhibits good activity.

[0026] The DNA molecules described above in this invention can also exist in the form of an "expression cassette." An "expression cassette" refers to a linear or circular nucleic acid molecule encompassing DNA and RNA sequences capable of directing the expression of specific nucleotide sequences in appropriate host cells. Generally, it includes a promoter effectively linked to the target nucleotide, optionally linked to a termination signal and / or other regulatory elements. The expression cassette may also include sequences required for the correct translation of the nucleotide sequence. The coding region typically encodes the target protein, but may also encode target functional RNA in the sense or antisense direction, such as antisense RNA or untranslated RNA. Expression cassettes containing the target polynucleotide sequence can be chimeric, meaning that at least one of its components is heterologous to at least one of its other components.

[0027] According to a typical embodiment of the present invention, a recombinant plasmid is provided. The recombinant plasmid contains any of the aforementioned DNA molecules. The DNA molecules in the recombinant plasmid are positioned at appropriate locations within the plasmid, enabling the DNA molecules to be correctly and smoothly replicated, transcribed, or expressed.

[0028] Although the present invention uses the qualifier "contains" when defining the aforementioned DNA molecule, this does not mean that other sequences unrelated to its function can be arbitrarily added to both ends of the DNA sequence. Those skilled in the art will know that, in order to meet the requirements of recombination operations, it is necessary to add suitable restriction endonuclease cleavage sites to both ends of the DNA sequence, or to add additional start codons, stop codons, etc. Therefore, using a closed-form description to define it will not truly cover these situations.

[0029] The term "plasmid" as used in this invention includes any plasmid, granule, bacteriophage, or Agrobacterium binary nucleic acid molecule in double-stranded or single-stranded linear or circular form, preferably a recombinant expression plasmid. It can be a prokaryotic or eukaryotic expression plasmid, but prokaryotic expression plasmids are preferred. In some embodiments, any suitable vector can be used, such as cloning vectors like pUC18 and pUC19, or prokaryotic expression vectors like pET21 and pETite. This invention preferentially uses pETite as the vector. The host cell of the vector can be a prokaryotic cell, including Escherichia coli, or a eukaryotic cell, including yeast cells and Pichia pastoris cells. This invention preferentially uses HI-Control BL21(DE3) as the expression host cell.

[0030] According to a typical embodiment of the present invention, a method for producing nicotinamide mononucleotide is provided, comprising the step of catalyzing a reaction between nicotinamide ribose and ATP using nicotinamide ribokinase, wherein the nicotinamide ribokinase is any of the nicotinamide ribokinase mutants described above. This method can be a biocatalytic process or a fermentation process.

[0031] The aforementioned biocatalytic process for preparing nicotinamide mononucleotide (NMN) specifically refers to the process of converting a biocatalytic substrate into NMN. The bioenzyme used is the nicotinamide ribokinase mutant of this invention, or a combination of the nicotinamide ribokinase mutant of this invention and one or more other enzymes. The substrate can be nicotinamide ribose and ATP, or a precursor substance capable of being converted into nicotinamide ribose or ATP. For example: NMN is prepared using nicotinamide ribose and ATP as raw materials under the catalysis of the nicotinamide ribokinase mutant of this invention; NMN is prepared using nicotinamide, phosphoribosyl pyrophosphate (PRPP), and ATP as raw materials under the catalysis of the nicotinamide ribokinase mutant of this invention and nicotinamide phosphoribosyltransferase; NMN is prepared using nicotinamide, AMP, and pyrophosphate or their salts as raw materials under the catalysis of the nicotinamide ribokinase mutant of this invention and adenine phosphoribosyltransferase. Preferably, the nicotinamide ribokinase mutant of this invention is used in the form of enzyme solution, lyophilized enzyme powder, enzyme-containing cells, immobilized enzyme, or immobilized enzyme-containing cells. Preferably, the temperature of the catalytic reaction is 37°C; the pH of the catalytic reaction is 7.0–8.0°C; and the concentration of the catalytic reaction substrate nicotinamide ribose is 5–60 mM.

[0032] The beneficial effects of the present invention will be further illustrated below with reference to embodiments.

[0033] The nicotinamide ribokinase used in the following examples is obtained by artificially designed site-directed mutation of the original nicotinamide ribokinase nucleotide sequence as shown in SEQ ID NO:1.

[0034] Example 1

[0035] Construction of recombinant vectors and expression strains containing the parental nicotinamide ribokinase (NRK1) gene

[0036] The gene sequence of nicotinamide ribokinase (NRK1) from human parents (GenBank accession number: AY611480.1) published in the gene bank was first codon optimized, resulting in a gene length of 597 bases and a sequence as SEQ ID NO:3. Then, the full-length NRK1 sequence nrk1 was artificially synthesized (by a commercial synthesis company). The synthesized product was recombined into the multiple cloning site of the linearized plasmid pETite C-His using the Gibson self-assembly method, yielding the recombinant plasmid pETite-nrk1. This plasmid was transformed into Turbo chemicompetent cloning strain (NEB) and plated on LB agar medium containing 30 μg / ml kanamycin, and cultured overnight at 37°C. Plasmids were extracted from pETite-nrk 1 / Turbo positive clones using a plasmid extraction kit (NEB Monarch) and sequenced using primers pETite-T7Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5).DNA sequencing confirmed the nucleotide sequence of the parent nicotinamide ribokinase as shown in SEQ ID NO: 3 and its amino acid sequence as shown in SEQ ID NO: 2.

[0037] Example 2

[0038] Preparation of nicotinamide ribokinase mutants

[0039] The PCR amplification reaction system consisted of: 25 μL 2×KOD One PCR Master Mix (Toyobo), 1.5 μL 10 μmol / L upstream primer, 1.5 μL 10 μmol / L downstream primer, 1.0 μL 1.0 ng / μL pETite-nrk1 plasmid, and 21 μL ddH2O. The PCR amplification conditions were: denaturation at 98℃ for 10 seconds; annealing at 55℃ for 5 seconds; extension at 68℃ for 15 seconds, for 30 cycles, and storage at 4℃.

[0040] 1. Preparation of I6V mutant

[0041] Using the following primer pairs: nrk1 / I6V-F: ATGAAGACCTTTATTGTTGGTATTAGCGGTGTTACCAA (SEQ ID NO: 6), nrk1 / I6V-R: CCAACAATAAAGGTCTTCATATGTATATCTCCTTCT (SEQ ID NO: 7), and with the plasmid pETite-nrk1 constructed in Example 1 as a template, the nrk1 / I6V mutant gene was amplified by high-fidelity PCR using the above-described PCR amplification reaction system and conditions. The PCR product was recovered from the gel using a DNA gel extraction kit to obtain the recombinant plasmid pETite-nrk1 / I6V. This plasmid was then transformed into Turbo chemicompetent cloning cells, plated on LB agar medium containing 50 μg / ml kanamycin, and incubated overnight at 37°C. Plasmids from the pETite-nrk1 / I6V / Turbo positive clone strain were extracted using a plasmid extraction kit, and sequencing was performed using primers pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7 Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). DNA sequencing confirmed the nucleotide sequence as that of the amide ribokinase mutant I6V.

[0042] 2. Preparation of S34H mutant

[0043] Using the following primer pairs: nrk1 / S34H-F: 5'AGCGTTATTCATCAGGATGATTTTTTCAAACCGG 3' (SEQ ID NO: 8), nrk1 / S34H-R: 5'TCATCCTGATGAATAACGCTACAATTCGGCA 3' (SEQ ID NO: 9), and with the plasmid pETite-nrk1 constructed in Example 1 as a template, the nrk1 / S34H mutant gene was amplified by high-fidelity PCR using the above-described PCR amplification reaction system and conditions. The PCR product was recovered from the gel using a DNA gel extraction kit to obtain the recombinant plasmid pETite-nrk1 / S34H. This plasmid was then transformed into Turbo chemicompetent cloning cells, plated on LB agar medium containing 50 μg / ml kanamycin, and incubated overnight at 37°C. Plasmids from the pETite-nrk1 / S34H / Turbo positive clone strain were extracted using a plasmid extraction kit, and sequencing was performed using primers pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7 Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). DNA sequencing confirmed the nucleotide sequence as that of the amide ribokinase mutant S34H.

[0044] 3. Preparation of T47V mutant

[0045] Using the following primer pairs: nrk1 / T47V-F: 5'GATAAAAATGGTTTCCTGCAGTATGATGTTC 3' (SEQ ID NO: 10), nrk1 / T47V-R: 5'TGCAGGAAACCATTTTTATCAACTTCGATTTCGCTTTCCGGTTTG3' (SEQ ID NO: 11), and with the plasmid pETite-nrk1 constructed in Example 1 as a template, the nrk1 / T47V mutant gene was amplified by high-fidelity PCR using the above-described PCR amplification reaction system and conditions. The PCR product was recovered from the gel using a DNA gel extraction kit to obtain the recombinant plasmid pETite-nrk1 / T47V. This plasmid was then transformed into Turbo chemicompetent cloning strains and plated onto LB agar medium containing 50 μg / ml kanamycin, and cultured overnight at 37°C. Plasmids from the pETite-nrk1 / T47V / Turbo positive clone strain were extracted using a plasmid extraction kit. Sequencing was performed using primers pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7 Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). DNA sequencing confirmed the nucleotide sequence as that of the amide ribokinase mutant T47V.

[0046] 4. Preparation of the I6V+S34H+T47V mutant

[0047] The full-length sequence of nrk1 / I6V+S34H+T47V was artificially synthesized (by a commercially available company). The synthesized product was recombined into the multiple cloning site of the linearized plasmid pETite C-His using the Gibson self-assembly method, yielding the recombinant plasmid pETite-nrk1 / I6V+S34H+T47V. This plasmid was transformed into Turbo chemicompetent clones (NEB) and plated on LB agar containing 30 μg / ml kanamycin, then incubated overnight at 37°C. The plasmid was extracted from the pETite-nrk1 / I6V+S34H+T47V / Turbo positive clone using a plasmid extraction kit (NEB Monarch), and sequenced using primers pETite-T7 Forward: ACGACTCACTATAGGGTGTGAG (SEQ ID NO: 4) and pETite-T7 Reverse: CTCAAGACCCGTTTAGAGGC (SEQ ID NO: 5). DNA sequencing confirmed the nucleotide sequence as the amide ribokinase mutant I6V+S34H+T47V.

[0048] Example 3

[0049] Enzyme induction, expression, isolation, and purification

[0050] The plasmid pETite-nrk1 containing the parental nicotinamide ribokinase gene and the plasmids pETite-nrk1 / I6V, pETite-nrk1 / S34, pETite-nrk1 / T47V, and nrk1 / I6V+S34H+T47V containing the nicotinamide ribokinase mutant gene were transformed into E. coli HI-Control BL21(DE3) cells, respectively, and cultured at 37°C for approximately 20 hours on LB agar plates containing 50 μg / mL kanamycin. Single colonies were picked and inoculated into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and cultured at 37°C for approximately 12–18 hours (OD). 600 The primary seed culture was obtained by diluting the sample to 0.4 g / mL. The primary seed culture was then inoculated into 100 mL of LB liquid medium containing 50 μg / mL kanamycin at a volume ratio of 2:100 and incubated at 37°C and 270 rpm for 3–4 hours (OD200). 600 The secondary seed culture was obtained by diluting the sample to 0.4%. The secondary seed culture was then inoculated into 500 mL of LB liquid medium containing 50 μg / mL kanamycin at a volume ratio of 2:100 and incubated at 37°C and 200 rpm for 3–4 hours (OD200). 600 The culture was then cooled to 18°C, and isopropyl-β-D-thiogalactoside (IPTG) was added as an inducer. The cells were incubated at 18°C ​​and 200 rpm for 16–18 hours to induce NRK1 and its mutant enzyme expression. The cells were collected by centrifugation at 5000 rpm for 5 minutes at 4°C. The cells were then resuspended in 6 volumes of PBS buffer, washed, and centrifuged again at 5000 rpm for 5 minutes at 4°C. The cells were then resuspended in 50 mM Tris-HCl and 100 mM NaCl buffer (pH 7.4) to obtain a cell suspension. The cells were then disrupted using a high-pressure cryogenic cell disruptor to release the target enzyme. The cell suspension was centrifuged at 12000 rpm for 10 minutes at 4°C, and the supernatant was collected. This yielded the crude enzyme solution of parental nicotinamide ribokinase NRK1 and its series of mutants, which was used for further enzyme isolation, purification, and activity assay.

[0051] Example 4

[0052] Enzyme isolation and purification

[0053] The crude enzyme solution was filtered through a 0.45 μM pore size filter membrane and purified by a protein purifier and nickel affinity chromatography. Equilibration buffer A consisted of 50 mM Tris-HCl, 100 mM NaCl, and 5 mM imidiazole, pH 7.4; elution buffer B was a 100% imidazole solution, pH 7.4. At a flow rate of 2.0 mL / min, the elution peak of 30% B elution buffer (i.e., 0.3 M imidazole) was the target protein peak. The imidazole was then removed using a HiPrep 26 / 10 Desalting column to obtain highly pure NRK1 and its mutant enzymes. Enzyme activity was determined using nicotinamide ribose and ATP as substrates. Enzyme concentration was determined using the Bradford method with BSA (bovine serum albumin) as the protein standard.

[0054] Example 5

[0055] Enzyme activity assay

[0056] The following solutions were prepared using 50 mM Tris-HCl + 50 mM NaCl at pH 7.4: 60 mM nicotinamide ribose, 60 mM ATP, and 100 mM MgCl2. 60 μL of each solution was mixed thoroughly, and then 20 μL of the purified enzyme solution obtained in Example 4 was added. The reaction was carried out at 37°C and 240 rpm with shaking for 10 minutes. The reaction was terminated by adding 800 μL of acetonitrile-water solution (V:V = 8:2). The concentrations of the substrate nicotinamide ribose and the product nicotinamide mononucleotide were determined using a Waters Alliance e2695 / 2998 high-performance liquid chromatography (HPLC) system with an Atlantis HILIC Silica column (Waters, 4.6 × 150 mm, 5.0 μm particle size). The injection volume was 2 μL, and the flow rate was 1.2 mL / min. -1 The column temperature was 28℃. Mobile phase A consisted of 0.1% (g / L) ammonium acetate + 5% (V:V) acetonitrile-water, and mobile phase B consisted of acetonitrile. The gradient elution curves were 0-7 min 75% B, 7.1-13 min 40% B, and 13.1-24 min 75% B. The flow rate was 1.2 mL / min, and the detection wavelength was 254 nm. The retention times of nicotinamide ribose, ATP, and nicotinamide mononucleotide were approximately 4.0 min, 7.3 min, and 9.0 min, respectively. The contents of the substrate nicotinamide ribose and the product nicotinamide mononucleotide (NMN) in the reaction solution were determined by high-performance liquid chromatography (HPLC). Enzyme activity was calculated, such as... Figure 1As shown. One unit (U) of nicotinamide ribokinase activity is defined as the amount of nicotinamide ribokinase required to convert 1.0 μmol of nicotinamide ribose per minute at 37°C and pH 7.4. Enzyme activity is expressed as specific activity (U / mg), which is the number of enzyme activity units per milligram of protein. The enzyme activity of mutants was also detected using the same method. The results are shown below. Figure 1 The images show the enzyme catalytic activities of these mutants I6V, S34H, T47V, and I6V+S34H+T47V using nicotinamide ribose and ATP as substrates. These activities are 2.5, 3.6, 2.3, and 5.5 times that of their parents, respectively, and they can be applied in the process of preparing nicotinamide mononucleotides.

[0057] Example 6

[0058] The reaction process for synthesizing NMN: taking the catalytic reaction of the NRK1 mutant I6V+S34H+T47V as an example.

[0059] In a reaction vessel, 60 mL of 60 mM nicotinamide ribose substrate solution, 105 mL of 0.1 M ATP (excess), and 15 mL of 0.8 M MgCl2 (excess) were added. Then, 20 mL of a 3.0 mg / mL mutant I6V+S34H+T47V purified enzyme solution was added, and the mixture was thoroughly mixed. The pH was maintained at 7.0–7.4, and the reaction temperature was controlled at 37℃ and the rotation speed at 180 rpm. 0.92 g of nicotinamide ribose was added at 30 min and 60 min, respectively. After 100 min of reaction, according to HPLC analysis, the conversion rate of the substrate nicotinamide ribose was 72%, yielding a crude NMN product solution (containing 54 mM NMN). After centrifugation, purification, and drying, NMN was obtained.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A nicotinamide riboside kinase mutant with increased activity, characterized in that, The nicotinamide riboside kinase mutant is a mutant sequence of the amino acid sequence shown in SEQ ID NO: 2, and the mutation site is I6V, S34H, T47V or I6V+S34H+T47V.

2. A DNA molecule, characterized in that, The DNA molecule encodes the nicotinamide riboside kinase mutant of claim 1.

3. A recombinant plasmid, characterized in that, The recombinant plasmid is linked with the DNA molecule of claim 2.

4. The recombinant plasmid of claim 3, wherein, The carrier plasmid of the recombinant plasmid is pBR327, pAT153, pUC18, pUC19, pET21 or pETite.

5. The recombinant plasmid of claim 4, wherein, The carrier plasmid of the recombinant plasmid is pETite.

6. A host cell, characterized in that, The host cell contains the recombinant plasmid of any one of claims 3 to 5.

7. The host cell of claim 6, wherein, The host cell includes a prokaryotic cell or a eukaryotic cell.

8. The host cell of claim 7, wherein, The prokaryotic cell is BL21 (DE3) Escherichia coli.

9. The host cell of claim 7, wherein, The eukaryotic cell is a yeast cell.

10. A method for producing nicotinamide mononucleotide, comprising catalyzing a reaction of nicotinamide ribose and ATP, or a precursor capable of being converted into nicotinamide ribose or ATP, by nicotinamide riboside kinase, characterized in that, The nicotinamide riboside kinase is the nicotinamide riboside kinase mutant of claim 1.

11. The method of claim 10, wherein, The catalytic reaction includes: using nicotinamide riboside and ATP as raw materials, preparing NMN under the catalysis of the nicotinamide riboside kinase mutant; Or, using nicotinamide, ribose phosphate pyrophosphate and ATP as raw materials, preparing NMN under the catalysis of the nicotinamide riboside kinase mutant and nicotinamide ribose phosphate transferase; Or, using nicotinamide, AMP and pyrophosphate or its salt as raw materials, preparing NMN under the catalysis of the nicotinamide riboside kinase mutant and adenine ribose phosphate transferase.

12. The method of claim 11, wherein, The use form of the nicotinamide riboside kinase mutant is a purified enzyme solution, an enzyme freeze-dried powder, an enzyme-containing cell, an immobilized enzyme or an immobilized enzyme-containing cell.

13. The method of claim 11, wherein, The temperature of the catalytic reaction is 36-37℃.

14. The method of claim 11, wherein, The pH of the catalytic reaction is 7.0-8.0℃.

15. The method of claim 11, wherein, The concentration of the catalytic reaction substrate nicotinamide riboside is 5-60 mM.

16. The host cell of claim 9, wherein, The eukaryotic cell is a Pichia pastoris cell.

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