WO2018023210A1 - Procédé de préparation de mononucléotide de nicotinamide - Google Patents

Procédé de préparation de mononucléotide de nicotinamide Download PDF

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WO2018023210A1
WO2018023210A1 PCT/CN2016/092461 CN2016092461W WO2018023210A1 WO 2018023210 A1 WO2018023210 A1 WO 2018023210A1 CN 2016092461 W CN2016092461 W CN 2016092461W WO 2018023210 A1 WO2018023210 A1 WO 2018023210A1
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nicotinamide
reaction
prset
phosphoribosyltransferase
amino acid
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PCT/CN2016/092461
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Chinese (zh)
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傅荣昭
张琦
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邦泰生物工程(深圳)有限公司
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Priority to PCT/CN2016/092461 priority Critical patent/WO2018023210A1/fr
Priority to CN201680003973.8A priority patent/CN107889505B/zh
Publication of WO2018023210A1 publication Critical patent/WO2018023210A1/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 mononucleotides after long-term experiments, which are characterized by: nicotinamide, pyrophosphoric acid or its salt and inosinic acid or The salt is used as a raw material, and reacts under the catalysis of nicotinamide phosphoribosyltransferase, hypoxanthine phosphoribosyltransferase and xanthine oxidase to obtain a nicotinamide mononucleotide.
  • the EC numbers of the enzymes used in the above methods are: nicotinamide phosphoribosyltransferase EC 2.4.2.12, hypoxanthine phosphoribosyltransferase EC 2.4.2.8, xanthine oxidation Enzyme EC 1.17.3.2.
  • 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.
  • the inventors found through comparison experiments that under the same conditions, the yield of NMN in the above-mentioned biocatalytic reaction in the absence of xanthine oxidase is greatly reduced, and the reason is mainly This is because the intermediate product formed during the reaction inhibits the formation of NMN, and the addition of xanthine oxidase and the removal of the intermediate by hydrazine eliminates its inhibitory effect on NMN.
  • 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.
  • 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 carrier can be selected from epoxy type LX-3000, silica, activated carbon and glass beads. And macroporous poly N-aminoethyl acrylamide-polyethylene and the like.
  • the pyrophosphate salt is a sodium salt of pyrophosphate, including sodium pyrophosphate, disodium pyrophosphate.
  • the inosinate is a sodium salt of inosinic acid, including sodium inosinate, disodium inosinate.
  • 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 ruthenium conversion rate 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+ , Zn 2+ and sodium hydrogen sulfite.
  • the reaction is carried out in Tris-HCl buffer.
  • the concentration of the nicotinamide is 1-100 mM, and the concentration of the pyrophosphoric acid or a salt thereof is 1-50 mM.
  • the concentration of the inosinic acid or a salt thereof is from 1 to 50 mM. .
  • the molar ratio of nicotinamide, pyrophosphoric acid or its salt, inosinic acid or its salt in the raw material is 1-3:1-2:
  • the conversion rate calculated by the substrate inosine or its salt can be 80% - 10
  • 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):
  • the above nicotinamide phosphoribosyltransferase is a site-directed mutagenesis of the gene of the parent nicotinamide phosphoribosyltransferase of the nucleotide sequence of Meiothermus ruber DSM 1279, which is represented by 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 is associated with the amino acid sequence set forth in SEQ ID NO: 2.
  • the ratio has at least one mutation selected from at least one of the following positions: No. 180, No. 182, No. 231, No. 298, No. 338, and No. 377.
  • 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 calculated by the substrate inosine or its salt is up to 100%, which is the most environmentally friendly and pollution-free in the preparation method of the current nicotinamide mononucleotide, suitable for large-scale industrial production and low in price.
  • PRPP the conversion rate calculated by the substrate inosine or its salt is up to 100%, which is the most environmentally friendly and pollution-free in the preparation method of the current nicotinamide mononucleotide, 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 [0031] One-step feeding method:
  • the raw materials are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-100 mM of nicotinamide, l-50 mM pyrophosphoric acid or a salt thereof, l-50 mM of inosinic acid or a salt thereof, -30 mM MgCl 2 , l-30 mM ZnCl 2
  • nicotinamide phosphoribosyltransferase 1-100 g/L substrate solution hypoxanthine phosphoribosyltransferase 1-100 g/L Substrate solution, xanthine oxidase 1-100 g / L substrate solution.
  • stirring well the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled to 30-50 ° C, and the pH was maintained at 6.5-8.5.
  • a crude nicotinamide mononucleotide solution is obtained, which is filtered, purified and dried to obtain a finished nicotinamide single nucleotide.
  • Step by step feeding method :
  • a substrate solution having a composition of 1-50 mM pyrophosphoric acid or a salt thereof, l-50 mM inosinic acid or a salt thereof, l-30 mM MgCl 2 , l
  • the pH was adjusted to 6.5-8.5 by using -30 mM ZnCl 2 , l-50 mM sodium hydrogen sulfite, and 50-100 mM Tris-HCl buffer.
  • the following catalytic enzyme was then added to the substrate solution: hypoxanthine phosphoribosyltransferase 1-100 g/L substrate solution, xanthine oxidase 1-100 g/L substrate solution.
  • stirring was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled to 30-50 ° C, and the pH was 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, except for the nicotinamide phosphoribosyltransferase mutant, 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, hypoxanthine phosphoribosyltransferase, and xanthine oxidase are commercially available lyophilized powders directly purchased from the market.
  • Embodiment 1 Preparation of Nicotinamide Mononucleotide
  • a substrate solution was added to the reaction vessel containing 1 mM nicotinamide, 1 mM pyrophosphate, 1 mM sodium inosinate, 1 mM MgCl 2 , 1 mM ZnCl 2 , 1 mM sodium bisulfite, 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, hypoxanthine phosphoribosyltransferase lg/L substrate solution, Xanthine oxidase lg/L substrate solution.
  • a substrate solution was added to the reaction vessel containing 20 mM sodium pyrophosphate, 10 mM disodium inosinate, 10 mM MgCl 2 , 10 mM ZnCl 2 , 20 mM sodium bisulfite, and 70 mM Tris-HCl buffer. Liquid, adjust the pH to 7.0-7.5.
  • the following catalytic enzymes were then added to the substrate solution: hypoxanthine phosphoribosyltransferase 30 g/L substrate solution, xanthine oxidase 35 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 35 ° C, and the pH was maintained at 7.0-7.5.
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 30 mM of nicotinamide, 10 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 NMN10 mM) is obtained, which is filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
  • a substrate solution was added to the reaction vessel containing 30 mM disodium pyrophosphate, 30 mM sodium inosinate, 20 mM MgCl 2 , 20 mM ZnCl 2 , 40 mM sodium bisulfite, and 100 mM Tris-HCl buffer. Liquid, adjust the pH to 7.5-8.0.
  • the following catalytic enzymes were then added to the substrate solution: hypoxanthine phosphoribosyltransferase 40 g/L substrate solution, xanthine oxidase 50 g/L substrate solution. After stirring evenly, the reaction is carried out, and the reaction is carried out. Stirring was continued during the process (stirring speed 50 rpm), the reaction temperature was controlled at 40 ° C, and the pH was maintained at 7.5-8.0.
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 70 mM of nicotinamide, 20 mM of MgCl 2 , and 100 mM of Tris-HCl buffer were added to the reaction solution.
  • nicotinamide phosphoribosyltransferase 40g / L substrate solution continue to react after stirring, continue stirring during the reaction process (stirring speed 50rpm), control the reaction temperature is 30-50 ° C, maintain the pH value of 7.5-8.0, and then After 5 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN 27.3 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.
  • a substrate solution was added to the reaction vessel containing 100 mM nicotinamide, 50 mM sodium pyrophosphate, 50 mM inosine, 30 mM MgCl 2 , 30 mM ZnCl 2
  • the mixture was filtered through a filter bag and washed with a washing enzyme buffer for 5-6 times to obtain immobilized nicotinamide phosphoribosyltransferase, immobilized hypoxanthine phosphoribosyltransferase, and immobilized xanthine oxidase.
  • the substrate solution was added to the reaction kettle, containing 60 mM of nicotinamide, 30 mM disodium pyrophosphate, 20 mM
  • the inosine disodium, 20 mM MgCl 2 , 20 mM ZnCl 2 , 35 mM sodium bisulfite, and 100 mM Tris-HCl buffer were adjusted to pH 7.0-7.5.
  • the preparation process of artificially induced site-directed mutagenesis of nicotinamide phosphoribosyltransferase is roughly as follows: First, construct a vector plasmid containing a parent nicotinamide phosphoribosyltransferase gene, and then set a site-directed mutagenesis. 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 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 a volume of 50 ⁇ l with sterile water.
  • the PCR amplification reaction conditions were: 95 ° C for 3 minutes, 35 cycles of 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 72 ° C for 5 minutes.
  • coliBL21 and a clone having nicotinamide phosphoribosyltransferase activity was selected on a Luriabroth (LB) plate (containing 50 mg/L kanamycin), and a plasmid was extracted from the clone.
  • the DNA of pRSET-F180A was confirmed by DN A sequencing to confirm that the introduced point mutation was correct.
  • the amino acid sequence of the F180A mutant was mutated from Phe (F) to Ala (A) at the 180th position, and its amino acid sequence is shown in SEQ ID NO: 3, as compared with the parent amino acid sequence shown in SEQ ID NO: 2.
  • 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 compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
  • 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 as shown in SEQ ID NO: 2.
  • E231Q-F 5' CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3'
  • E231Q-R 5' GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 3'
  • High-fidelity PCR amplification of the E231Q 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 1), plasmid pRSET-E231Q was obtained.
  • 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 as shown 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.
  • D298N mutant Using the following primer pair D298N-F: 5' GTTGTTATCCGTCCGAATTCTGGTGACCCGCCG 3' and D298N-R: 5' CGGCGGGTCACCAGAATTCGGACGGATAACAAC 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 PC R expansion Increasing the reaction conditions The high-fidelity PCR amplification of the D298N mutant gene was carried out 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 details).
  • the 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.
  • the DNA of the plasmid pRSET-D298N was extracted, and the point mutation introduced was confirmed by DNA sequencing.
  • the amino acid sequence of the D298N mutant was mutated from Asp (D) to Asn (N) at position 298 as 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. Corresponding to the parent amino acid sequence as shown in SEQ ID NO: 2. The amino acid sequence of the D377A mutant was mutated from Asp (D) to Ala at the 377th position.
  • 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
  • the PCR and amplification conditions were subjected to high-fidelity PCR amplification of the E231Q/D338E 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 (specifically Referring to Example 6 Part I), plasmid pRSET-21 was obtained.
  • 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).
  • 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, sieved on a Luria broth (LB) plate (containing 50 mg/L kanamycin). A clone having nicotinamide phosphoribosyltransferase activity was selected, and the DN A of the plasmid pRSET-23 was extracted from the clone, and the point mutation introduced was confirmed by DNA sequencing. Compared with the parent amino acid sequence shown in SEQ ID NO: 2, 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 was used as a template, and the above PCR amplification reaction system and PCR amplification reaction conditions were subjected to high fidelity PCR.
  • the E231Q/D338E/D377E 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 (refer to the first part of Example 6 for details). Plasmid pRSET-31.
  • 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 is at position 231 by Glu (E) compared to the parent amino acid sequence set forth in SEQ ID NO: 2. Mutant to Gin (Q), from Asp (D) to Ala (A) at position 298, Asp (D) to Glu (E) at position 338, and Asp (at point 377) D) Mutation to Glu (E).
  • E231Q ⁇ pRSET-D298A, pRSET-D298N, pRSET-D298E, pRSET-D338N, pRSET-D338E, pRSET-D377A, pRSET-D377N, pRSET-D377E, pRSET-21, pRSET-22, pRSET-23, pRSET-31 and pRSET-41 was transformed into competent bacterial cells E. coli BL21, respectively, and 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 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.
  • the substrate solution was added to the reaction vessel, containing 60 mM nicotinamide, 30 mM disodium pyrophosphate, 20 mM disodium inosinate, 20 mM MgCl 2 , 20 mM ZnCl 2 , 35 mM sodium hydrogen sulfite, and lOOmM Tris-HCl buffer, adjusted to pH 7.0-7.5. Then, various enzymes for catalysis were added to the substrate solution, and the amounts of the various enzymes added 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 hypoxanthine phosphoribosyltransferase 10 g/L substrate solution, xanthine oxidase 20 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 19.8 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, acide pyrophosphorique ou un sel de celui-ci et de l'acide inosinique ou un sel de celui-ci réagissent sous les effets catalytiques de la nicotinamide phosphoribosyltransférase, de l'hypoxanthine phosphoribosyltransférase et de la xanthine oxydase de façon à obtenir un mononucléotide de nicotinamide.
PCT/CN2016/092461 2016-07-30 2016-07-30 Procédé de préparation de mononucléotide de nicotinamide WO2018023210A1 (fr)

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US11878027B2 (en) 2015-08-05 2024-01-23 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US11059847B2 (en) 2015-10-02 2021-07-13 Metro International Biotech, Llc Crystal forms of β-nicotinamide mononucleotide
US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
US11180521B2 (en) 2018-01-30 2021-11-23 Metro International Biotech, Llc Nicotinamide riboside analogs, pharmaceutical compositions, and uses thereof
US10618927B1 (en) 2019-03-22 2020-04-14 Metro International Biotech, Llc Compositions and methods for modulation of nicotinamide adenine dinucleotide
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
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
US11952396B1 (en) 2021-05-27 2024-04-09 Metro International Biotech, Llc Crystalline solids of nicotinic acid mononucleotide and esters thereof and methods of making and use
JP7111878B1 (ja) 2021-10-27 2022-08-02 旭化成ファーマ株式会社 ニコチンアミドモノヌクレオチドの製造方法
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JP2023065161A (ja) * 2021-10-27 2023-05-12 旭化成ファーマ株式会社 ニコチンアミドモノヌクレオチドの製造方法
US11959116B2 (en) 2021-10-27 2024-04-16 Asahi Kasei Pharma Corporation Method for producing nicotinamide mononucleotide

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