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WO2018023209A1 - Procédé de préparation de mononucléotide de nicotinamide - Google Patents

Procédé de préparation de mononucléotide de nicotinamide Download PDF

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Publication number
WO2018023209A1
WO2018023209A1 PCT/CN2016/092460 CN2016092460W WO2018023209A1 WO 2018023209 A1 WO2018023209 A1 WO 2018023209A1 CN 2016092460 W CN2016092460 W CN 2016092460W WO 2018023209 A1 WO2018023209 A1 WO 2018023209A1
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Prior art keywords
nicotinamide
prset
reaction
amino acid
acid sequence
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PCT/CN2016/092460
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Chinese (zh)
Inventor
傅荣昭
张琦
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Bontac Bio-Engineering (shenzhen) Co Ltd
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Bontac Bio-Engineering (shenzhen) Co Ltd
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Priority to CN201680003974.2A priority Critical patent/CN108026535B/zh
Priority to PCT/CN2016/092460 priority patent/WO2018023209A1/fr
Publication of WO2018023209A1 publication Critical patent/WO2018023209A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/455—Nicotinic acids, e.g. niacin; Derivatives thereof, e.g. esters, amides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes

Definitions

  • the present invention relates to the field of molecular biology and biotechnology, and more particularly to a method for preparing a nicotinamide mononucleotide using a biocatalytic technique.
  • Nicotinamide mononucleotide is a biochemical substance present in biological cells, which is transformed into a biological cell after being adenylated by nicotinamide nucleotide adenosyltransferase.
  • nicotinamide mononucleotides have many health care uses such as delaying aging, treating senile diseases such as Parkinson's, regulating insulin secretion, affecting mRNA expression, and the like, and more applications are being continuously developed. .
  • senile diseases such as Parkinson's
  • regulating insulin secretion affecting mRNA expression, and the like
  • mRNA expression affecting mRNA expression
  • the demand for nicotinamide mononucleotides is increasing.
  • the preparation method of NMN mainly includes the following three types: 1. yeast fermentation method; 2. chemical synthesis method; 3. biocatalysis method.
  • the chemical synthesis method has the disadvantages of high cost and the production of chiral compounds; and the NMN produced by the yeast fermentation method contains certain organic solvent residues; the biocatalytic method does not contain the solvent residue, and there is no chiral problem and is prepared.
  • NMN is the same as the same type in the body and has become the most green and environmentally friendly NMN preparation method.
  • the existing biocatalytic method for preparing NMN is generally based on nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate (PRPP), and Nicotinamide e phosphoribosyltransferase (abbreviated to Nampt). Preparation of NMN under catalysis.
  • PRPP nicotinamide and 5'-phosphoribosyl-1'-pyrophosphate
  • Nampt Nicotinamide e phosphoribosyltransferase
  • an object of the present invention is to provide a novel method for preparing NMN by using a biocatalytic technique, which is avoidable PRUse high-priced and limited-source PRPP as a substrate, which has the advantages of low price, environmental protection, pollution-free, and suitable for large-scale industrial production.
  • nicotinamide mononucleotide after long-term experimentation, characterized in that: nicotinamide, ATP and AMP are used as raw materials, and nicotinamide phosphate is used.
  • the reaction of ribose transferase, ribose phosphate pyrophosphate kinase and AMP nucleosidase is carried out to obtain a nicotinamide mononucleotide.
  • the EC numbers of the enzymes used in the above methods are: nicotinamide phosphoribosyltransferase EC 2.4.2.12, ribose phosphate pyrophosphate kinase EC 2.7.6.1, AMP nucleosidase EC 3.2.2.4.
  • the specific forms of the various enzymes used in the above methods include an enzyme solution, an enzyme lyophilized powder, an enzyme-containing cell, and various immobilized enzymes and immobilized enzyme-containing cells, which may be in the form of unpurified crude enzyme. It may also be in a partially purified or fully purified form.
  • an immobilized enzyme in the above method.
  • the immobilized enzyme is prepared by: diluting the enzyme to a protein content of 5-10 mg/ml with a washing enzyme buffer (0.02 M Tris-HCl/0.001 M EDTA, pH 7.0 solution), and then diluting the enzyme with PB solution (2.0 mol / L potassium dihydrogen phosphate, pH 7.5) was mixed in equal volume, then added to the enzyme immobilization carrier (50 mg enzyme / gram carrier), reacted at 25 ° C in a shaker (rotation speed 150 rpm) 20 small Inches.
  • an immobilized enzyme After the reaction is completed, it is filtered with a filter bag and washed with a washing enzyme buffer for 5-6 times to obtain an immobilized enzyme.
  • the enzyme-immobilized carriers epoxy type LX-3000, silica, activated carbon, glass beads, and macroporous poly-N-aminoethyl acrylamide-polyethylene can be used.
  • the reaction is carried out at a temperature of 30 to 50 ° C and a pH of 6.5 to 8.5.
  • the reaction has the highest conversion of hydrazine at a temperature of 35-45 ° C and a pH of 7.0-8.0.
  • the reaction is carried out in the presence of Mg 2+ and K + .
  • the reaction is carried out in Tris-HCl buffer.
  • the concentration of the nicotinamide is 1-150 mM
  • the concentration of the ATP is 1-50 mM
  • the concentration of the AM P is 1-100 mM.
  • the molar ratio of nicotinamide, ATP, and AMP in the raw material is 1-6:1:1-2. ATP can be fully reacted by placing the raw materials in this ratio, and the conversion rate is 80 ⁇ 3 ⁇ 4-100 ⁇ 3 ⁇ 4. Since the price of ATP is the highest among the three raw materials, this ratio can greatly reduce the production cost. More preferably, the molar ratio of nicotinamide, ATP, and AMP in the raw material is 3-4:1:1.5, and the conversion ratio calculated by the substrate ATP is 100%, and the cost is the lowest.
  • the nicotinamide mononucleotide crude product solution obtained after the completion of the reaction can be subjected to filtration, purification and drying treatment by a conventional technique known in the art, that is, a nicotinamide single nucleotide product can be obtained.
  • the nicotinamide phosphoribosyltransferase used in the above method is a protein of the following (a) or (b):
  • nicotinamide phosphoribosyltransferase is a site-directed mutagenesis of the gene of the parent nicotinamide phosphoribosyltransferase represented by the nucleotide sequence of Meiothermus ruber DSM 1279, as shown in SEQ ID NO: 1, after PCR amplification.
  • a series of highly catalytically active nicotinamide phosphoribosyltransferase mutants were obtained by inserting an appropriate vector and subsequently screening on LB+ kanamycin medium. The high catalytic activity of these mutants can greatly reduce industrial application organisms.
  • the cost of catalytic technology for the production of nicotinamide mononucleotides has high industrial application value.
  • the nicotinamide phosphoribosyltransferase has at least one mutation selected from at least one of the following positions compared to the amino acid sequence set forth in SEQ ID NO: 2: position 180, position 182 , 231rd, 298th, 338th and 377th.
  • the nicotinamide phosphoribosyltransferase has at least one of the following mutations: F180A, F180 W, A182Y, E231A, E231Q, D298A, D298N, D298E, D338N, D338E, D37 7A, D377N and D377E.
  • the method provided by the invention overcomes the defects of the chemical synthesis method and the yeast fermentation method, and successfully avoids the price and the source limitation.
  • the use of PRPP, the conversion rate of the method calculated by the substrate ATP is as high as 100%, and belongs to the preparation method of the current nicotinamide mononucleotide, which is the most environmentally friendly and pollution-free, suitable for large-scale industrial production and low in price.
  • the nicotinamide phosphoribosyltransferase used in the method provided by the present invention is a mutant obtained by artificially induced site-directed mutagenesis, and the enzyme activity of the mutant is greatly improved compared with the existing wild type.
  • the enzymatic activity assay uses nicotinamide and PRPP as substrates.
  • the enzyme has a catalytic activity of 1.2-6.9 times that of the parent. Such high catalytic activity allows it to be used as a crude enzyme without purification, or only a part of it.
  • the preparation method of the nicotinamide mononucleotide provided by the invention may be a one-step feeding method in which all raw materials and enzymes are added together, or a stepwise feeding method, and the one-step feeding method has simple operation and short reaction time. Advantages, step-by-step feeding method has the advantages of thorough reaction and high conversion rate.
  • the specific implementation process is as follows
  • the raw materials were prepared by dissolving each raw material in water, and the composition of the substrate solution was 1-150 mM of nicotinamide, 1-50 mM of ATP, 1-100 mM of AMP, and 1-30 mM of MgCl 2 .
  • Step by step feeding method :
  • the raw materials were prepared by dissolving each raw material in water, and the composition of the substrate solution was 1-50 mM ATP, 1-10 OmM AMP, l-30 mM MgCl 2 , l-20 mM KC1, and 50-100 mM. Tris-HCl buffer, adjusted to pH 6.5-8.5. The following catalytic enzymes were then added to the substrate solution: ribose phosphate pyrophosphate kinase 1-100 g/L substrate solution, AMP nucleosidase 1-100 g/L substrate solution. After stirring evenly, the reaction is carried out, stirring is continued during the reaction (stirring speed 50 rpm), the reaction temperature is controlled at 30-50 ° C, and the pH is maintained at 6.5-8.5.
  • reaction solution is separated, and 1-100 mM of nicotinamide and 1-30 mM of MgCl 2 are added to the reaction solution.
  • the enzyme used in the following examples is the parent nicotinamide phosphoribosyltransferase from the nucleotide sequence of Meiother mus ruber DSM 1279, as shown in SEQ ID NO: 1.
  • the remaining nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, and AMP nucleosidase are commercially available lyophilized powders directly purchased from the market.
  • the substrate solution was added to the reaction vessel, containing 1 mM nicotinamide, 1 mM ATP, 1 mM AMP, lm M MgCl 2 , 1 mM KC1, and 50 mM Tris-HCl buffer, adjusted to pH 6.5-7.0 .
  • various enzymes for catalysis are added to the substrate solution, and the amounts of the various enzymes are: nicotinamide phosphoribosyltransferase lg/L substrate solution, ribose phosphate pyrophosphate kinase lg/L substrate solution, AMP core Glycosidase lg/L substrate solution.
  • a substrate solution was added to the reaction vessel containing 20 mM ATP, 30 mM AMP, 15 mM MgCl 2
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 70 mM of nicotinamide, 15 mM of MgCl 2 , and 70 mM of Tris-HCl buffer were added to the reaction solution.
  • nicotinamide phosphoribosyltransferase 30g / L substrate solution stirring evenly, continue the reaction, stirring during the reaction (mixing speed 50rpm), control the reaction temperature is 35 ° C, maintain the pH of 7.0-7.5, and then react for 3h After that, a crude nicotinamide mononucleotide solution (containing NMN 20 mM) is obtained, which is filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
  • a substrate solution containing 40 mM ATP, 50 mM AMP, 20 mM MgCl 2 , 15 mM KCl, and 100 mM Tris-HCl buffer was added to the reaction vessel to adjust the pH to 7.5-8.0.
  • the following catalytic enzymes were then added to the substrate solution: ribose phosphate pyrophosphate kinase 70 g/L substrate solution, AMP nucleosidase 70 g/L substrate solution.
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 100 mM of nicotinamide, 20 mM of MgCl 2 , and 100 mM of Tris-HCl buffer were added to the reaction solution.
  • nicotinamide phosphoribosyltransferase 70g / L substrate solution continue to react after stirring, continue stirring during the reaction (mixing speed 50rpm), control the reaction temperature is 40 ° C, maintain the pH value of 7.5-8.0, and then react for 5h After that, a crude nicotinamide mononucleotide solution (containing NMN38.5 mM) is obtained, which is filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
  • a substrate solution containing 150 mM of nicotinamide, 50 mM of ATP, 100 mM of AMP, 30 mM of MgCl 2 , 20 mM of KCl, and 100 mM of Tris-HCl buffer was added to the reaction vessel to adjust the pH to 8.0-8.5.
  • various enzymes for catalysis are added to the substrate solution, and the amounts of the various enzymes are: nicotinamide phosphoribosyltransferase 100 g/L substrate solution, ribose phosphate pyrophosphate kinase 100 g/L substrate solution, AMP core Glycosidase 100 g / L substrate solution.
  • a substrate solution containing 60 mM of nicotinamide, 10 mM of ATP, 20 mM of AMP, 15 mM of MgCl 2 , 15 mM of KCl and 100 mM of Tris-HCl buffer was added to the reaction vessel to adjust the pH to 7.0-7.5.
  • various enzymes for catalysis are added to the substrate solution, and the amounts of the various enzymes are as follows: immobilized nicotinamide phosphoribosyltransferase 10 g/L substrate solution, immobilized ribose phosphate pyrophosphate kinase 10 g/L substrate Solution, immobilized AMP nucleosidase 10 g / L substrate solution.
  • immobilized nicotinamide phosphoribosyltransferase 10 g/L substrate solution immobilized ribose phosphate pyrophosphate kinase 10 g/L substrate Solution
  • immobilized AMP nucleosidase 10 g / L substrate solution After stirring uniformly, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 37 ° C, and the pH was maintained at 7.0-7.5. After 3 hours of reaction, a crude nicotinamide mononucleo
  • the preparation process of the artificially induced site-directed mutagenesis nicotinamide phosphoribosyltransferase used in the method provided by the present invention is roughly as follows: First, a vector plasmid containing a parent nicotinamide phosphoribosyltransferase gene is constructed, and then a site-directed mutagenesis is set. The site and the amino acid species after mutation, and then synthesize appropriate primers, and use the vector plasmid containing the parent nicotinamide phosphoribosyltransferase gene as a template to PCR-amplify the DNA fragment, assemble the amplified DNA fragment, and fully expand the PCR amplification. Mutant gene.
  • the full-length mutant gene is then cloned into an appropriate vector and transformed into an appropriate host cell, and a positive clone having nicotinamide phosphoribosyltransferase activity is selected by culture. Finally, the plasmid DNA is extracted from the positive clone, and the DNA sequence analysis is performed to determine the introduced mutation. After the target fragment is inserted into the vector, the LB+ kanamycin medium is selected for screening, thereby obtaining a series of high catalytic activity. Nicotinamide phosphoribosyltransferase mutant.
  • any suitable vector may be employed, for example, it may be a prokaryotic expression vector such as pRSET and pES21, etc.; and may be a cloning vector such as pUC18/19 and pBluscript-SK.
  • pRSET-A is preferably used as a vector, and the host cell of the vector may be a prokaryotic cell including Escherichia coli, or a eukaryotic cell including Saccharomyces cerevisiae and P. pastoris.
  • the nucleotide sequence of the cloned parent nicotinamide phosphoribosyltransferase was determined by DNA sequencing as shown in SEQ ID NO: 1, and the amino acid sequence thereof is shown in SEQ ID NO: 2.
  • the PCR amplification reaction system is: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH 4 ) 2 SO 4 , 2 mM MgS0 4 , 0.1% Triton X-100, 50 mM dATP, 50 mM dTTP, 50 mM dCTP, 50 mM dGTP, 1.5 U Pfu DNA polymerase (Promega, USA), 20 ng DNA template, and 400 nM upstream primer and 400 nM downstream primer, adjusted to 50 ⁇ m with sterile water Rise.
  • the PCR amplification reaction conditions were: 95 ° C for 3 minutes, 35 cycles: 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C 3 Minutes, last 72 ° C for 5 minutes.
  • the plasmid pRSET-F180W was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-F180W was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the F180W mutant was mutated from Phe (F) to Trp (w) at the 180th position as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • the following primer pair A182Y-F 5'
  • the plasmid pRSET-nampt constructed in the first part was used as a template, and the A182Y mutant gene was amplified by high-fidelity PCR using the above PCR amplification reaction system and PCR amplification reaction conditions, separated by 1% agarose gel electrophoresis and recovered by commercial kit.
  • the product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain the plasmid pRSET-A182Y.
  • the plasmid pRSET-A182Y was transformed into competent bacterial cell E.
  • plasmid pRSET-A182Y was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the A182Y mutant was mutated from Ala (A) to Tyr (Y) at position 182 compared to the parent amino acid sequence set forth in SEQ ID NO: 2.
  • plasmid pRSET-E231A was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231A mutant was mutated from Glu (E) to Ala (A) at position 231 compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • E231Q-F 5' CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3'
  • E231Q-R 5' GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 3'
  • the plasmid pRSET-E231Q was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-E231Q was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231Q mutant was mutated from Glu (E) to Gin (Q) at position 231 as compared to the parent amino acid sequence set forth in SEQ ID NO: 2.
  • the plasmid pRSET-D298A was obtained.
  • the plasmid pRSET-D298A was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-D298A was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the D298A mutant was changed from Asp (D) to Ala (A) at the 298th position as compared with the parent amino acid sequence as shown in SEQ ID NO: 2.
  • the PCR amplification reaction was carried out using the plasmid pRSET-nampt constructed in the first part of Example 6 using the following plasmid pair D298N-F: 5' GTTGTTATCCGTCCGAATTCTGGTGACCCGCCG 3' and D298N-R: 5' CGGCGGGTCACCAGAATTCGGACGGATAACAAC 3' System and PC R amplification reaction conditions High-fidelity PCR amplification of the D298N mutant gene, separation by 1% agarose gel electrophoresis and recovery of the amplified product with a commercial kit, and then the amplification product was ligated with the vector pRSET-A (specific reference) Example 6 Part I), plasmid pRSET-D298N was obtained.
  • the plasmid pRSET-D298N was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. Extract the plasmid pRSET-D298N from D NA, the point mutation introduced by DNA sequencing was confirmed to be correct. The amino acid sequence of the D298N mutant was mutated from Asp (D) to Asn at position 298 compared to the parent amino acid sequence set forth in SEQ ID NO: 2.
  • D298E-R 5' GAACGGCGGGTCACCAGATTCCGGACGGATAACAAC 3', using the plasmid pRSET-nampt constructed in the first step of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification
  • the D298E mutant gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D298E.
  • the plasmid pRSET-D298E was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-D298E was extracted, and the point mutation introduced was confirmed by DN A sequencing.
  • the amino acid sequence of the D298E mutant was mutated from Asp (D) to Glu (E) at position 298 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • D338E-R 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D338E mutant
  • the gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D338E.
  • the plasmid pRSET-D338E was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-D338E was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the D338E mutant was mutated from Asp (D) to Glu (E) at position 338 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • D377A-R 5' GAATTTCTGGGTCGCACGGTGCGGGTG 3', using the plasmid pRSET-nampt constructed in the first step of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D377A
  • the mutant gene was separated by electrophoresis on a 1% agarose gel and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D377A.
  • the plasmid pRSET-D377A was transformed into competent bacterial cell E.
  • plasmid pRSET-D377A was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the D377A mutant was mutated from Asp (D) to Ala (A) at position 377 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • D377E-R 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-nampt constructed in the first part of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of D377E mutant
  • the gene was isolated by electrophoresis on a 1% agarose gel and the amplified product was recovered using a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET-D377E.
  • the plasmid pRSET-D377E was transformed into competent bacterial cell E.
  • D338E-R 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3', using the plasmid pRSET-E231Q constructed in the fifth subsection of Example 6, Part 2, as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high fidelity PCR
  • the E231Q/D338E mutant gene was amplified, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference to the first part of Example 6) to obtain a plasmid pRSET. -twenty one.
  • the plasmid pRSET-21 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-2 1 was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231Q/D338E mutant was mutated from Glu (E) to Gin (Q) at position 231 and by Asp (D) at position 338, compared to the parent amino acid sequence set forth in SEQ ID NO: 2. ) Mutation to Glu (E). [0089] 15. Preparation of E231Q/D377E mutant
  • D377E-R 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-E231Q constructed in the fifth subsection of the second part of Example 6 as a template, using the above PCR amplification reaction system and PC R amplification reaction conditions for high fidelity
  • the E231Q/D377E mutant gene was amplified by PCR, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (refer to the first part of Example 6 for specific reference) to obtain a plasmid. pRSET-22.
  • the plasmid pRSET-22 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-22 was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231Q/D377E mutant was mutated from Glu (E) to Gin (Q) at position 231 and by Asp (D) at position 377, compared to the parent amino acid sequence as shown in SEQ ID NO: 2. ) Mutation to Glu (E).
  • D377E-R 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', using the plasmid pRSET-D338E constructed in the 10th subsection of the second part of Example 6 as a template, using the above PCR amplification reaction system and P CR amplification reaction conditions for high fidelity
  • the D338E/D377E mutant gene was amplified by PCR, separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (refer to the first part of Example 6 for specific reference) to obtain a plasmid.
  • pRSET-23 5' CGAATTTCTGGGTTTCACGGTGCGGG 3'
  • the plasmid pRSET-23 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DN A of the plasmid pRSET-23 was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the D338E/D377E mutant was mutated from Asp (D) to G1 u (E) at position 338 and by Asp at position 377 ( D) Mutation to Glu (E).
  • D377E-R 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', the plasmid pRSET-21 constructed in the 14th subsection of the second part of Example 6 as a template, the above PCR amplification reaction system and PCR Amplification reaction conditions were carried out by high-fidelity PCR amplification of the E231Q/D338E/D377E mutant gene, which was separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and the amplified product was ligated with the vector pRSET-A (specific reference) Example 6 Part I), plasmid pRSET-31 was obtained.
  • the plasmid pRSET-31 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • the DNA of the plasmid pRSET-31 was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231Q/D338E/D377E mutant was mutated from Glu (E ) to Gin (Q) at position 231 and from Asp at position 338, compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • D Mutation to Glu (E), mutation from Asp (D) to Glu (E) at position 377.
  • the plasmid pRSET-31 constructed in the 17th subsection of the second part of Example 6 was used as a template, using the above PCR
  • the amplification reaction system and the PCR amplification reaction conditions were subjected to high-fidelity PCR amplification of the E231Q/D298A/D338E/D377E mutant gene, which was separated by 1% agarose gel electrophoresis and the amplified product was recovered by a commercial kit, and then the amplification product was used.
  • the vector pRSET-A was ligated (specifically with reference to the first part of Example 6) to obtain plasmid pRSET-41.
  • the plasmid pRSET-41 was transformed into competent bacterial cell E. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on a Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone.
  • LB Luria broth
  • the DNA of the plasmid pRSET-41 was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the E231Q/D298A/D338E/D377E mutant was mutated from Glu (E ) to Gin (Q) at position 231, at position 298, compared to the parent amino acid sequence set forth in SEQ ID NO: 2. It was mutated from Asp (D) to Ala (A), from Asp (D) to Glu (E) at position 338, and from Asp (D) to Glu (E) at position 377.
  • the bacterial cell E. coli BL21 was cultured on a Luria broth (LB) plate (containing 50 mg/L kanamycin) for 24 hours at 37 °C. Inoculate a single clone in 50 ml of LB liquid medium (including 50
  • nicotinamide mononucleotide (NMN) in the reaction solution was determined by high performance liquid chromatography (HPL C), and The specific enzyme activity of each enzyme was calculated, and the specific activity of the parent nicotinamide phosphoribosyltransferase was referred to as reference 100.
  • the relative activities of the parent and each mutant are shown in Table 1.
  • a substrate solution containing 60 mM of nicotinamide, 10 mM of ATP, 20 mM of AMP, 15 mM of MgCl 2 , 15 mM of KCl and 100 mM of Tris-HCl buffer was added to the reaction vessel to adjust the pH to 7.0-7.5.
  • Various enzymes for catalysis were added to the substrate solution, and the amounts of the various enzymes were as follows:
  • the supernatant protein solution of the nicotinamide phosphoribosyltransferase mutant (F180A) obtained in the third part of Example 6 was 10 ml/L.
  • Substrate solution ribose phosphate pyrophosphate kinase 15 g / L substrate solution, AMP nucleosidase 15 g / L substrate solution.
  • stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 37 ° C, and the pH was maintained at 7.0-7.5.
  • a crude nicotinamide mononucleotide solution (containing NMN 9.8 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.

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Abstract

L'invention concerne un procédé de préparation de mononucléotide de nicotinamide. Les matières premières de nicotinamide, ATP et AMP réagissent sous les effets catalytiques de la nicotinamide phosphoribosyltransférase, de la ribose phosphate pyrophosphokinase et de l'AMP nucléosidase de manière à obtenir un mononucléotide de nicotinamide.
PCT/CN2016/092460 2016-07-30 2016-07-30 Procédé de préparation de mononucléotide de nicotinamide Ceased WO2018023209A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
US10548913B2 (en) 2015-08-05 2020-02-04 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10618927B1 (en) 2019-03-22 2020-04-14 Metro International Biotech, Llc Compositions and methods for modulation of nicotinamide adenine dinucleotide
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US11180521B2 (en) 2018-01-30 2021-11-23 Metro International Biotech, Llc Nicotinamide riboside analogs, pharmaceutical compositions, and uses thereof
CN114164190A (zh) * 2021-10-12 2022-03-11 南宁邦尔克生物技术有限责任公司 一种生产烟酰胺单核苷酸的融合酶及其应用
CN115537439A (zh) * 2022-04-27 2022-12-30 长沙创新药物工业技术研究院有限公司 一种固定化酶制备β-烟酰胺单核苷酸的方法
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US11787830B2 (en) 2021-05-27 2023-10-17 Metro International Biotech, Llc Crystalline solids of nicotinic acid mononucleotide and esters thereof and methods of making and use
US11939348B2 (en) 2019-03-22 2024-03-26 Metro International Biotech, Llc Compositions comprising a phosphorus derivative of nicotinamide riboside and methods for modulation of nicotinamide adenine dinucleotide
US11959116B2 (en) 2021-10-27 2024-04-16 Asahi Kasei Pharma Corporation Method for producing nicotinamide mononucleotide

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112646851B (zh) * 2019-10-10 2023-03-28 安徽古特生物科技有限公司 一种酶法快速制备β-烟酰胺单核苷酸的方法
CN116004489B (zh) * 2022-07-04 2024-11-08 华熙生物科技股份有限公司 一种生产nmn的重组大肠杆菌及其应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246803A1 (en) * 2004-09-20 2009-10-01 Washington University Nad biosynthesis systems
WO2015069860A1 (fr) * 2013-11-06 2015-05-14 President And Fellows Of Harvard College Production biologique de précurseurs et d'analogues de nad

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101601679B (zh) * 2009-03-17 2011-08-10 中国人民解放军第二军医大学 一种烟酰胺单核苷酸的应用
CN104814974A (zh) * 2015-03-16 2015-08-05 邦泰生物工程(深圳)有限公司 烟酰胺单核苷酸在制备抗衰老药物或保健品的应用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090246803A1 (en) * 2004-09-20 2009-10-01 Washington University Nad biosynthesis systems
WO2015069860A1 (fr) * 2013-11-06 2015-05-14 President And Fellows Of Harvard College Production biologique de précurseurs et d'analogues de nad

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE IUBMB "EC 2.7.6. 1", XP055460573, Database accession no. EC 2.7.6. 1 *
DATABASE IUBMB 31 December 1961 (1961-12-31), "EC . 2. 2. 4", Database accession no. EC . 2. 2. 4 *

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