WO2018023207A1 - Method for preparing nicotinamide mononucleotide - Google Patents

Method for preparing nicotinamide mononucleotide Download PDF

Info

Publication number
WO2018023207A1
WO2018023207A1 PCT/CN2016/092457 CN2016092457W WO2018023207A1 WO 2018023207 A1 WO2018023207 A1 WO 2018023207A1 CN 2016092457 W CN2016092457 W CN 2016092457W WO 2018023207 A1 WO2018023207 A1 WO 2018023207A1
Authority
WO
WIPO (PCT)
Prior art keywords
nicotinamide
prset
reaction
amino acid
acid sequence
Prior art date
Application number
PCT/CN2016/092457
Other languages
French (fr)
Chinese (zh)
Inventor
傅荣昭
张琦
Original Assignee
邦泰生物工程(深圳)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 邦泰生物工程(深圳)有限公司 filed Critical 邦泰生物工程(深圳)有限公司
Priority to CN201680003960.0A priority Critical patent/CN107889504B/en
Priority to PCT/CN2016/092457 priority patent/WO2018023207A1/en
Publication of WO2018023207A1 publication Critical patent/WO2018023207A1/en

Links

Classifications

    • 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, which is characterized in that: nicotinamide, ATP and xylose are used as raw materials in nicotinamide.
  • the reaction is carried out under the catalysis of phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase. 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, ribose-5-phosphate Isomerase EC 5.3.1.6, ribulose-3-phosphate isomerase EC 5.1.3.1, xylulokinase EC 2.7.1.17, xylose isomerase EC 5.3.1.5.
  • 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 is carried out at a temperature of 35-45 ° C and a pH of 7.0-8.0. The highest.
  • the reaction is carried out in the presence of Mg 2+ , K + and Zn 2+ .
  • the reaction is carried out in Tris-HCl buffer.
  • the concentration of the nicotinamide is 1 to 150 mM
  • the concentration of the ATP is 1 to 50 mM
  • the concentration of the xylose is 1 to 50 mM.
  • the molar ratio of nicotinamide, ATP, and xylose in the raw material is 1-4:1:1-4, and the ATP is fully reacted according to the ratio of the raw materials, 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, xylose in the raw material is 1.5:1:1.5, the conversion rate 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 are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-150 mM nicotinamide, l-50 mM ATP, l-50 mM xylose, l-30 mM MgCl 2
  • nicotinamide phosphoribosyltransferase 1-100 g/L substrate solution ribose phosphate pyrophosphate kinase 1-100 g/L substrate Solution
  • ribose-5-phosphate isomerase 1-100 g/L substrate solution ribulose-3-phosphate isomerase 1-100 g/L substrate solution
  • xylulose kinase 1-100 g/L substrate solution xylose isomerase l-100g / L substrate solution.
  • Step by step feeding method :
  • the raw materials are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-50 mM ATP, 1-50 mM xylose, l-30 mM MgCl 2 , l-20 mM KC1, and 50- 100 mM Tris-HCl buffer was 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, ribose-5-phosphate isomerase 1-100 g/L substrate solution, ribulose-3-phosphate Isomerase 1-100 g/L substrate solution, xylulose kinase 1-100 g/L substrate solution, xylose isomerase 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 nicotinamide, l-50 mM ZnCl 2 is added to the reaction solution.
  • the enzyme used in the following examples, except 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 sugar isomerases are enzyme lyophilized powders purchased directly from the market.
  • Nicotinamide Mononucleotide A substrate solution containing 1 mM nicotinamide, 1 mM ATP, 1 mM xylose, lm M MgCl 2 , 1 mM KC1, 1 mM ZnCl 2 was added to the reaction vessel.
  • Tris-HCl buffer adjust the pH to 6.5-7.0.
  • various enzymes for catalysis are: nicotinamide phosphoribosyltransferase lg/L substrate solution, ribose phosphate pyrophosphate kinase lg/L substrate solution, ribose-5-phosphate isomerase Lg/L substrate solution, ribulose-3-phosphate isomerase lg/L substrate solution, xylulose kinase lg/L substrate solution, xylose isomerase lg/L substrate solution.
  • a substrate solution containing 30 mM ATP, 30 mM xylose, 20 mM MgCl 2 , 10 mM KC1, 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 ribose phosphate pyrophosphate kinase 6g / L substrate solution, ribose-5-phosphate isomerase 10g / L substrate solution, ribulose - 3-phosphate isomerase l lg / L substrate Solution, xylulose kinase 10 g / L substrate solution, xylose isomerase 10 g / L substrate solution.
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 60 mM of nicotinamide, 30 mM of ZnCl 2 , and 100 mM of Tris-HCl buffer were added to the reaction solution.
  • nicotinamide phosphoribosyltransferase 15g / L substrate solution continue to react after stirring, continue stirring during the reaction (mixing speed 50rpm), control the reaction temperature is 35 ° C, maintain the pH value of 7.5-8.0, and then react for 4h After that, a crude nicotinamide mononucleotide solution (containing NMN 12 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 25 mM ATP, 50 mM xylose, 15 mM MgCl 2
  • reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 50 mM of nicotinamide, 20 mM of ZnCl 2 , and 70 mM of Tris-HCl buffer were added to the reaction solution.
  • nicotinamide phosphoribosyltransferase 50g / 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 3h After that, a crude nicotinamide mononucleotide solution (containing NMN12.4 mM) was obtained, which was filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
  • the substrate solution was added to the reaction vessel, containing 150 mM nicotinamide, 50 mM ATP, 50 mM xylose, 30 mM MgCl 2 , 20 mM KC1, 10 mM ZnCl 2 and 100 mM Tris-HCl buffer. 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, ribose- 5-phosphate isomerase 100g/L substrate solution, ribulose-3-phosphate isomerase 100g/L substrate solution, xylulose kinase 100g/L substrate solution, xylose isomerase 100g/L bottom Solution.
  • reaction 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 immobilized nicotinamide phosphoribosyltransferase, immobilized ribose phosphate pyrophosphate kinase, immobilized ribose-5-phosphate isomerase, Immobilized ribulose-3-phosphate isomerase, immobilized xylulokinase, and immobilized xylose isomerase.
  • a substrate solution containing 100 mM of nicotinamide, 30 mM of ATP, 30 mM of xylose, 12 mM of MgCl 2 , 10 mM of KC1, 10 mM of ZnCl 2 , and 100 mM of Tris-HCl buffer was added to the reaction vessel. Adjust the pH to 7.0-7.5.
  • various enzymes for catalysis are added, and the amounts of various enzymes added are: immobilized nicotinamide phosphoribosyltransferase 15 g/L substrate solution, immobilized ribose phosphate pyrophosphate kinase 6 g/L substrate solution, immobilized ribose- 5-phosphate isomerase 10g/L substrate solution, immobilized ribulose-3-phosphate isomerase l lg/L substrate solution, immobilized xylulokinase 10g/L substrate solution, immobilized xylose Isomerase 10 g / L substrate solution.
  • 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 preferred as the vector
  • 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 of 95 ° C for 50 seconds, 52 ° C for 30 seconds, and 72 ° C for 3 minutes, and finally 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 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.
  • the plasmid pRSET-nampt constructed in the first part was used as a template, and the E231A 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-E231A.
  • the plasmid pRSET-E231A was transformed into competent bacterial cell E.
  • 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 set forth 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 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.
  • 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 as shown 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 at position 298 (E
  • 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
  • the high-fidelity PCR amplification of the D377A mutant gene was performed 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-D377A was obtained.
  • 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).
  • 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).
  • 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.
  • LB Luria broth
  • 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 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 Amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of E231Q/D298A/D338E/D377E mutant gene, 1% agarose gel
  • the amplified product was isolated by swimming and recovered with 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 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.
  • 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.
  • 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
  • a substrate solution containing 30 mM nicotinamide, 20 mM ATP, 30 mM xylose, 12 mM MgCl 2 , 10 mM KC1, 10 mM ZnCl 2 , and 100 mM Tris-HCl buffer was added to the reaction vessel. Adjust the pH to 7.0-7.5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Urology & Nephrology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Hematology (AREA)
  • Analytical Chemistry (AREA)
  • Epidemiology (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Food Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Saccharide Compounds (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Disclosed is a method for preparing nicotinamide mononucleotide. Raw materials nicotinamide, ATP and xylose react under the catalytic effects of nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphokinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase so as to obtain nicotinamide mononucleotide.

Description

一种制备烟酰胺单核苷酸的方法  Method for preparing nicotinamide mononucleotide
技术领域  Technical field
[0001] 本发明涉及分子生物学与生物技术领域， 特别涉及一种利用生物催化技术制备 烟酰胺单核苷酸的方法。  [0001] 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.
背景技术  Background technique
[0002] 烟酰胺单核苷酸 （Nicotinamide mononucleotide, 缩写成 NMN) 是生物细胞内 存在的一种生化物质， 它在被烟酰胺核苷酸腺苷转移酶腺苷化后即转化成生物 细胞所赖以生存的重要物质烟酰胺腺嘌呤二核苷酸 （NAD， 又称辅酶 I) ， 并且 同吋直接参与体内腺苷转移， 其在生物细胞内的水平直接影响到 NAD的浓度， 在生物细胞能量生成中扮演着重要角色， 并且对人体无危害。  [0002] Nicotinamide mononucleotide (NMN) is a biochemical substance present in biological cells, which is transformed into a biological cell after being adenylated by nicotinamide nucleotide adenosyltransferase. The important substance that depends on the survival of nicotinamide adenine dinucleotide (NAD, also known as coenzyme I), and homologous directly involved in adenosine transfer in vivo, its level in biological cells directly affects the concentration of NAD, in biological cells It plays an important role in energy generation and is harmless to the human body.
[0003] 截至目前， 人们已经发现烟酰胺单核苷酸具有诸如延缓衰老、 治疗帕金森等老 年病、 调节胰岛素分泌、 影响 mRNA的表达等诸多医疗保健用途， 更多的用途还 在不断研发出来。 随着人们对烟酰胺单核苷酸药用及保健效果认知的增加， 以 及作为一种反应底物在化工方面的广泛应用， 市场上对烟酰胺单核苷酸的需求 量与日俱增。 [0003] Up to now, it has been found that 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. . With the increasing awareness of the medicinal and health effects of nicotinamide mononucleotides and the widespread use of chemicals as a reaction substrate, the demand for nicotinamide mononucleotides is increasing.
[0004] 目前， NMN的制备方法主要包括以下三种： 1、 酵母菌发酵法； 2、 化学合成 法； 3、 生物催化法。 其中， 化学合成法具有成本较高且产生手性化合物的缺点 ； 而酵母菌发酵法生产的 NMN含一定有机溶剂残留； 生物催化法因不含机溶剂 残留， 也不存在手性问题且制备的 NMN与机体内的同型而成为目前最绿色环保 无公害的 NMN的制备方法。 现有的制备 NMN的生物催化法一般是以烟酰胺和 5'- 磷酸核糖基 -1'-焦磷酸 （PRPP) 为底物， 在烟酰胺磷酸核糖转移酶 （Nicotinamid e phosphoribosyltransferase, 缩写成 Nampt) 的催化下制备 NMN。 但是， 因 PRPP 的市场价格较高且来源受限， 导致该生物催化法的生产成本较高， 严重制约了 其应用和发展。  [0004] At present, the preparation method of NMN mainly includes the following three types: 1. yeast fermentation method; 2. chemical synthesis method; 3. biocatalysis method. Among them, 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. However, due to the high market price and limited source of PRPP, the production cost of the biocatalytic method is high, which seriously restricts its application and development.
[0005] 因此， 有必要幵发一种无需使用 PRPP作为底物的利用生物催化技术制备 NMN 的新方法。 技术问题 [0005] Therefore, it is necessary to develop a new method for preparing NMN using biocatalysis technology without using PRPP as a substrate. technical problem
[0006] 针对上述背景技术中提到的现有的烟酰胺单核苷酸的制备方法存在的诸多问题 ， 本发明的目的在于提供一种利用生物催化技术制备 NMN的新方法， 该方法可 避幵使用价格较高且来源受限的 PRPP作为底物， 具有价格低廉、 绿色环保无公 害、 适宜大规模工业化生产等优点。  [0006] In view of the problems in the preparation method of the existing nicotinamide mononucleotide mentioned in the above background art, 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.
问题的解决方案  Problem solution
技术解决方案  Technical solution
[0007] 为实验上述目的， 发明人经过长期大量的实验摸索， 终于幵发出一种制备烟酰 胺单核苷酸的方法， 其特征在于： 以烟酰胺、 ATP和木糖为原料， 在烟酰胺磷酸 核糖转移酶、 核糖磷酸焦磷酸激酶、 核糖 -5-磷酸异构酶、 核酮糖 - 3-磷酸异构酶 、 木酮糖激酶以及木糖异构酶的催化作用下发生反应， 制得烟酰胺单核苷酸。  [0007] In order to test the above object, the inventors have finally developed a method for preparing nicotinamide mononucleotide after long-term experimentation, which is characterized in that: nicotinamide, ATP and xylose are used as raw materials in nicotinamide. The reaction is carried out under the catalysis of phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase. Nicotinamide mononucleotide.
[0008] 根据酶的国际***命名法， 上述方法中所使用的酶的 EC编号分别为： 烟酰胺 磷酸核糖转移酶 EC 2.4.2.12， 核糖磷酸焦磷酸激酶 EC 2.7.6.1， 核糖 -5-磷酸异构 酶 EC 5.3.1.6， 核酮糖 -3-磷酸异构酶 EC 5.1.3.1， 木酮糖激酶 EC2.7.1.17， 木糖异 构酶 EC 5.3.1.5。  [0008] According to the international system nomenclature of enzymes, 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, ribose-5-phosphate Isomerase EC 5.3.1.6, ribulose-3-phosphate isomerase EC 5.1.3.1, xylulokinase EC 2.7.1.17, xylose isomerase EC 5.3.1.5.
[0009] 上述方法中所使用的各种酶的具体存在形式包括酶液、 酶冻干粉、 含酶细胞以 及各种固定化酶和固定化含酶细胞， 可以是未经纯化的粗酶形式， 也可以是经 部分纯化或完全纯化的形式。  [0009] 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.
[0010] 为提高所使用酶的稳定性和重复利用率， 以更好地完成上述催化反应并更进一 步降低成本， 上述方法中优选使用固定化酶。 该固定化酶的制备方法大致为： 用洗酶缓冲液 （0.02M Tris-HCl/0.001M EDTA， pH7.0溶液） 将酶稀释至蛋白含 量为 5-10mg/ml， 再将酶稀释液与 PB溶液 （2.0mol/L磷酸二氢钾， pH7.5) 等体积 混合， 然后加入酶固定化载体 （50毫克酶 /克载体） ， 于摇床 （转速 150rpm) 中 2 5°C反应 20小吋。 反应完成后用滤袋过滤， 用洗酶缓冲液清洗 5-6次， 即得固定化 酶。 其中的酶固定化载体可以选用环氧型 LX-3000、 二氧化硅、 活性炭、 玻璃珠 以及大孔型聚 N-氨乙基丙烯酰胺 -聚乙烯等。  [0010] In order to improve the stability and recyclability of the enzyme to be used, in order to better accomplish the above catalytic reaction and further reduce the cost, it is preferred to use 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. 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. Among the enzyme-immobilized carriers, epoxy type LX-3000, silica, activated carbon, glass beads, and macroporous poly-N-aminoethyl acrylamide-polyethylene can be used.
[0011] 优选地， 所述反应在温度为 30-50°C， pH值为 6.5-8.5的条件下进行。 Preferably, the reaction is carried out at a temperature of 30 to 50 ° C and a pH of 6.5 to 8.5.
[0012] 更优选地， 所述反应在温度为 35-45°C， pH值为 7.0-8.0的条件下进行吋转化率 最高。 More preferably, the reaction is carried out at a temperature of 35-45 ° C and a pH of 7.0-8.0. The highest.
[0013] 优选地， 所述反应在 Mg 2+、 K +和 Zn 2+存在的条件下进行。 Preferably, the reaction is carried out in the presence of Mg 2+ , K + and Zn 2+ .
[0014] 优选地， 所述反应在 Tris-HCl缓冲液中进行。 Preferably, the reaction is carried out in Tris-HCl buffer.
[0015] 优选地， 所述烟酰胺的浓度为 l-150mM， 所述 ATP的浓度为 l-50mM， 所述木 糖的浓度为 l-50mM。  Preferably, the concentration of the nicotinamide is 1 to 150 mM, the concentration of the ATP is 1 to 50 mM, and the concentration of the xylose is 1 to 50 mM.
[0016] 优选地， 所述原料中烟酰胺、 ATP、 木糖的摩尔比为 1-4:1:1-4， 按照此种配比 投放原料可使 ATP得到充分反应， 其转化率为 80<¾-100<¾。 因为在三种原料中， 以 ATP的售价为最高， 故而此种配比可较大程度地降低生产成本。 更优选地， 所 述原料中烟酰胺、 ATP、 木糖的摩尔比为 1.5:1:1.5， 反应以底物 ATP计算的转化 率为 100%， 且成本最低。  [0016] Preferably, the molar ratio of nicotinamide, ATP, and xylose in the raw material is 1-4:1:1-4, and the ATP is fully reacted according to the ratio of the raw materials, 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, xylose in the raw material is 1.5:1:1.5, the conversion rate calculated by the substrate ATP is 100%, and the cost is the lowest.
[0017] 反应完成后得到的烟酰胺单核苷酸粗产品溶液可采用本领域已知的常规技术手 段进行过滤、 纯化和干燥处理， 即得烟酰胺单核苷酸成品。  [0017] 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.
[0018] 优选地， 上述方法中所使用的烟酰胺磷酸核糖转移酶为如下 （a) 或 （b) 的蛋 白质：  Preferably, the nicotinamide phosphoribosyltransferase used in the above method is a protein of the following (a) or (b):
[0019] (a) 其氨基酸序列如 SEQ ID NO: 3所示的蛋白质，  (a) a protein having the amino acid sequence of SEQ ID NO: 3,
[0020] (b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个氨基酸并 且以烟酰胺和 PRPP为底物具有比氨基酸序列如 SEQ ID NO: 2所示的亲本高的烟 酰胺磷酸核糖转移酶催化活性的由 ) 衍生的蛋白质。  (b) having one or more amino acids substituted, deleted or added in the (a) defined amino acid sequence and having a nicotinamide and PRPP as a substrate having a higher amino acid sequence than the parent represented by SEQ ID NO: 2. The nicotinamide phosphoribosyltransferase catalytically active protein derived from).
[0021] 上述烟酰胺磷酸核糖转移酶是发明人对来自 Meiothermus ruber DSM 1279的核 苷酸序列如 SEQ ID NO: 1所示的亲本烟酰胺磷酸核糖转移酶的基因进行定点突 变， PCR扩增后***适当的载体， 随后在 LB+卡那霉素培养基上筛选而获得的一 系列具有高催化活性的烟酰胺磷酸核糖转移酶突变体， 这些突变体的高催化活 性可极大地降低工业上应用生物催化技术生产烟酰胺单核苷酸的成本， 具有较 高的工业应用价值。  The above-described 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.
[0022] 优选地， 所述烟酰胺磷酸核糖转移酶与如 SEQ ID NO: 2所示的氨基酸序列相 比在选自至少一个下述位点处具有至少一个突变： 第 180位、 第 182位、 第 231位 、 第 298位、 第 338位以及第 377位。  Preferably, 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.
[0023] 更优选地， 所述烟酰胺磷酸核糖转移酶具有至少一个下述突变： F180A、 F180 W、 A182Y、 E231A、 E231Q、 D298A、 D298N、 D298E、 D338N、 D338E、 D37 7A、 D377N以及 D377E。 More preferably, 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.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0024] 1、 与现有的烟酰胺单核苷酸的制备方法相比， 本发明提供的方法既克服了化 学合成法和酵母菌发酵法的缺陷， 又成功避免了价格昂贵且来源受限的 PRPP的 使用， 该方法以底物 ATP计算的转化率高达 100%， 属于现今烟酰胺单核苷酸的 制备方法中最为绿色环保无公害、 适宜大规模工业化生产且价格低廉的一种。  [0024] 1. Compared with the preparation method of the existing nicotinamide mononucleotide, 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.
[0025] 2、 本发明提供的方法中使用的烟酰胺磷酸核糖转移酶是一种经人工诱导定点 突变获得的突变体， 与现有的野生型相比， 该突变体的酶活力大大提高， 经酶 活测定， 以烟酰胺和 PRPP为底物， 该突变体的酶催化活性是亲本的 1.2-6.9倍， 如此高的催化活性使其可以未经纯化以粗酶形式使用， 或者只须部分纯化， 这 就使得应用本发明提供的烟酰胺磷酸核糖转移酶突变体催化生产 NMN的成本大 大降低， 具有较高的市场竞争力， 能够满足将 NMN的生物催化方法应用于大规 模工业化生产的需求。  [0025] 2. 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. Purification, which makes the cost of catalyzing the production of NMN by using the nicotinamide phosphoribosyltransferase mutant provided by the present invention greatly reduced, has high market competitiveness, and can meet the demand for applying the biocatalytic method of NMN to large-scale industrial production. .
本发明的实施方式 Embodiments of the invention
[0026] 下面结合具体实施例对本发明做进一步的详细说明， 以下实施例是对本发明的 解释， 本发明并不局限于以下实施例， 实施例中未注明具体条件者， 按常规条 件或制造商建议的条件进行。  The present invention will be further described in detail below with reference to the specific embodiments. The following examples are illustrative of the invention. The invention is not limited to the following examples, and the specific conditions are not indicated in the examples, according to conventional conditions or manufacturing. The conditions suggested by the business are carried out.
[0027] 本发明提供的烟酰胺单核苷酸的制备方法可以是将所有原料及酶一并加入的一 步投料方式也可以是分步投料方式， 一步投料方式具有操作简单、 反应吋间短 的优点， 分步投料方式具有反应彻底、 转化率高的优点， 其具体实施过程如下  [0027] 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
[0028] 一步投料方式： [0028] One-step feeding method:
[0029] 将各原料溶解于水中配制底物溶液， 该底物溶液的组成为 l-150mM的烟酰胺、 l-50mM的 ATP、 l-50mM的木糖、 l-30mM的 MgCl 2 [0029] The raw materials are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-150 mM nicotinamide, l-50 mM ATP, l-50 mM xylose, l-30 mM MgCl 2
、 l-20mM的 KC1、 1-lOmM的 ZnCl 2 以及 50-100mM的 Tris-HCl缓冲液， 调 pH至 6.5-8.5。 然后向底物溶液中加入催化 用的各种酶， 各种酶的加入量分别为： 烟酰胺磷酸核糖转移酶 l-100g/L底物溶液 ， 核糖磷酸焦磷酸激酶 l-100g/L底物溶液， 核糖 -5-磷酸异构酶 l-100g/L底物溶液 ， 核酮糖 - 3-磷酸异构酶 l-100g/L底物溶液， 木酮糖激酶 l-100g/L底物溶液， 木糖 异构酶 l-100g/L底物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速 度 50rpm) ， 控制反应温度为 30-50°C， 维持 pH值为 6.5-8.5。 反应 l-8h后即得烟酰 胺单核苷酸粗产品溶液， 再经过滤、 纯化、 干燥后即得烟酰胺单核苷酸成品。 , l-20mM KC1, 1-lOmM ZnCl 2 And 50-100 mM Tris-HCl buffer, adjust the pH to 6.5-8.5. Then, various enzymes for catalysis are added to the substrate solution, and the amounts of the various enzymes are: nicotinamide phosphoribosyltransferase 1-100 g/L substrate solution, ribose phosphate pyrophosphate kinase 1-100 g/L substrate Solution, ribose-5-phosphate isomerase 1-100 g/L substrate solution, ribulose-3-phosphate isomerase 1-100 g/L substrate solution, xylulose kinase 1-100 g/L substrate solution , xylose isomerase l-100g / 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 to 30-50 ° C, and the pH was maintained at 6.5-8.5. After 1-8 hours of reaction, a crude nicotinamide mononucleotide solution is obtained, which is filtered, purified and dried to obtain a finished nicotinamide single nucleotide.
[0030] 分步投料方式： [0030] Step by step feeding method:
[0031] 将各原料溶解于水中配制底物溶液， 该底物溶液的组成为 l-50mM的 ATP、 1-50 mM的木糖、 l-30mM的 MgCl 2、 l-20mM的 KC1以及 50-100mM的 Tris-HCl缓冲液 ， 调 pH至 6.5-8.5。 然后向底物溶液中加入以下催化用酶： 核糖磷酸焦磷酸激酶 1- 100g/L底物溶液， 核糖 -5-磷酸异构酶 l-100g/L底物溶液， 核酮糖- 3-磷酸异构酶 1- 100g/L底物溶液， 木酮糖激酶 1- 100g/L底物溶液， 木糖异构酶 1- 1 00g/L底物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速度 50rpm ) ， 控制反应温度为 30-50°C， 维持 pH值为 6.5-8.5。 [0031] The raw materials are prepared by dissolving each raw material in water, and the composition of the substrate solution is 1-50 mM ATP, 1-50 mM xylose, l-30 mM MgCl 2 , l-20 mM KC1, and 50- 100 mM Tris-HCl buffer was 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, ribose-5-phosphate isomerase 1-100 g/L substrate solution, ribulose-3-phosphate Isomerase 1-100 g/L substrate solution, xylulose kinase 1-100 g/L substrate solution, xylose isomerase 1-100 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 to 30-50 ° C, and the pH was maintained at 6.5-8.5.
[0032] 待上述反应进行 l-8h后， 分离出反应液， 并向反应液中加入 1-lOOmM的烟酰胺 、 l-50mM的 ZnCl 2 [0032] After the above reaction is carried out for 1-8 h, the reaction solution is separated, and 1-100 mM nicotinamide, l-50 mM ZnCl 2 is added to the reaction solution.
、 50- lOOmM的 Tris-HCl缓冲液以及烟酰胺磷酸核糖转移酶 1- 100g/L底物溶液， 搅 拌均匀后继续反应， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制反应温度为 30-50°C, 维持 pH值为 6.5-8.5， 再反应 l-8h后即得烟酰胺单核苷酸粗产品溶液， 再经过滤、 纯化、 干燥后即得烟酰胺单核苷酸成品。  50- lOOmM Tris-HCl buffer and nicotinamide phosphoribosyltransferase 1-100g/L substrate solution, stir evenly, continue the reaction, continue stirring during the reaction (stirring speed 50rpm), control reaction temperature is 30-50 °C, maintain the pH value of 6.5-8.5, and then react for l-8h to obtain the crude nicotinamide mononucleotide solution, and then filter, purify and dry to obtain the finished nicotinamide single nucleotide.
[0033] 以下实施例中所使用的酶， 除烟酰胺磷酸核糖转移酶突变体是从来自 Meiother mus ruber DSM 1279的核苷酸序列如 SEQ ID NO: 1所示的亲本烟酰胺磷酸核糖 转移酶经人工诱导定点突变获得的之外， 其余烟酰胺磷酸核糖转移酶、 核糖磷 酸焦磷酸激酶、 核糖 -5-磷酸异构酶、 核酮糖 - 3-磷酸异构酶、 木酮糖激酶以及木 糖异构酶均是从市场上直接购入的酶冻干粉。 The enzyme used in the following examples, except 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. In addition to artificially induced site-directed mutagenesis, the remaining nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate isomerase, ribulose-3-phosphate isomerase, xylulokinase, and wood The sugar isomerases are enzyme lyophilized powders purchased directly from the market.
[0034] 实施例 1  Embodiment 1
[0035] 烟酰胺单核苷酸的制备 [0036] 向反应釜中加入底物溶液， 含 ImM的烟酰胺、 ImM的 ATP、 ImM的木糖、 lm M的 MgCl 2、 ImM的 KC1、 ImM的 ZnCl 2 Preparation of Nicotinamide Mononucleotide [0036] A substrate solution containing 1 mM nicotinamide, 1 mM ATP, 1 mM xylose, lm M MgCl 2 , 1 mM KC1, 1 mM ZnCl 2 was added to the reaction vessel.
以及 50mM的 Tris-HCl缓冲液， 调 pH至 6.5-7.0。 然后加入催化用的各种酶， 各种 酶的加入量为： 烟酰胺磷酸核糖转移酶 lg/L底物溶液， 核糖磷酸焦磷酸激酶 lg/L 底物溶液， 核糖 -5-磷酸异构酶 lg/L底物溶液， 核酮糖 - 3-磷酸异构酶 lg/L底物溶 液， 木酮糖激酶 lg/L底物溶液， 木糖异构酶 lg/L底物溶液。 搅拌均匀后进行反应 ， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制反应温度为 30°C， 维持 pH值为 6.5-7.0。 反应 lh后即得烟酰胺单核苷酸粗产品溶液 （含 NMN0.5mM) ， 再经过 滤、 纯化、 干燥后即得烟酰胺单核苷酸。  And 50 mM Tris-HCl buffer, adjust the pH to 6.5-7.0. Then add various enzymes for catalysis. The amounts of various enzymes added are: nicotinamide phosphoribosyltransferase lg/L substrate solution, ribose phosphate pyrophosphate kinase lg/L substrate solution, ribose-5-phosphate isomerase Lg/L substrate solution, ribulose-3-phosphate isomerase lg/L substrate solution, xylulose kinase lg/L substrate solution, xylose isomerase lg/L substrate solution. After stirring well, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 30 ° C, and the pH was maintained at 6.5-7.0. After 1 h of reaction, a crude nicotinamide mononucleotide solution (containing NMN 0.5 mM) was obtained, which was filtered, purified and dried to obtain a nicotinamide mononucleotide.
[0037] 实施例 2  Example 2
[0038] 烟酰胺单核苷酸的制备  Preparation of Nicotinamide Mononucleotide
[0039] 向反应釜中加入底物溶液， 含 30mM的 ATP、 30mM的木糖、 20mM的 MgCl 2 、 10mM的 KC1以及 lOOmM的 Tris-HCl缓冲液， 调 pH至 7.5-8.0。 然后加入以下催 化用酶： 核糖磷酸焦磷酸激酶 6g/L底物溶液， 核糖 -5-磷酸异构酶 10g/L底物溶液 ， 核酮糖 - 3-磷酸异构酶 l lg/L底物溶液， 木酮糖激酶 10g/L底物溶液， 木糖异构 酶 10g/L底物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速度 50rp m) ， 控制反应温度为 35°C， 维持 pH值为 7.5-8.0。 [0039] A substrate solution containing 30 mM ATP, 30 mM xylose, 20 mM MgCl 2 , 10 mM KC1, and 100 mM Tris-HCl buffer was added to the reaction vessel to adjust the pH to 7.5-8.0. Then add the following catalytic enzymes: ribose phosphate pyrophosphate kinase 6g / L substrate solution, ribose-5-phosphate isomerase 10g / L substrate solution, ribulose - 3-phosphate isomerase l lg / L substrate Solution, xylulose kinase 10 g / L substrate solution, xylose isomerase 10 g / L substrate solution. After stirring uniformly, the reaction was carried out, stirring was continued during the reaction (stirring speed 50 rp m), the reaction temperature was controlled at 35 ° C, and the pH was maintained at 7.5-8.0.
[0040] 待上述反应进行 4h后， 分离出反应液， 并将反应液送入另一反应釜中， 再向反 应液中加入 60mM的烟酰胺、 30mM的 ZnCl 2、 lOOmM的 Tris-HCl缓冲液以及烟酰 胺磷酸核糖转移酶 15g/L底物溶液， 搅拌均匀后继续反应， 反应过程中持续搅拌 (搅拌速度 50rpm) ， 控制反应温度为 35°C， 维持 pH值为 7.5-8.0， 再反应 4h后即 得烟酰胺单核苷酸粗产品溶液 （含 NMN12mM) ， 再经过滤、 纯化、 干燥后即 得烟酰胺单核苷酸成品。 [0040] After the reaction was carried out for 4 hours, the reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 60 mM of nicotinamide, 30 mM of ZnCl 2 , and 100 mM of Tris-HCl buffer were added to the reaction solution. And nicotinamide phosphoribosyltransferase 15g / L substrate solution, continue to react after stirring, continue stirring during the reaction (mixing speed 50rpm), control the reaction temperature is 35 ° C, maintain the pH value of 7.5-8.0, and then react for 4h After that, a crude nicotinamide mononucleotide solution (containing NMN 12 mM) is obtained, which is filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
[0041] 实施例 3  Embodiment 3
[0042] 烟酰胺单核苷酸的制备  Preparation of Nicotinamide Mononucleotide
[0043] 向反应釜中加入底物溶液， 含 25mM的 ATP、 50mM的木糖、 15mM的 MgCl 2 [0043] A substrate solution was added to the reaction vessel containing 25 mM ATP, 50 mM xylose, 15 mM MgCl 2
、 10mM的 KC1以及 70mM的 Tris-HCl缓冲液， 调 pH至 7.0-7.5。 然后加入以下催化 用酶： 核糖磷酸焦磷酸激酶 20g/L底物溶液， 核糖 -5-磷酸异构酶 35g/L底物溶液， 核酮糖-10 mM KC1 and 70 mM Tris-HCl buffer were adjusted to pH 7.0-7.5. Then add the following catalytic enzymes: ribose phosphate pyrophosphate kinase 20g / L substrate solution, ribose-5-phosphate isomerase 35g / L substrate solution, Ribulose -
3-磷酸异构酶 35g/L底物溶液， 木酮糖激酶 35g/L底物溶液， 木糖异构酶 30g/L底 物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制 反应温度为 40°C， 维持 pH值为 7.0-7.5。 3-phosphate isomerase 35 g/L substrate solution, xylulose kinase 35 g/L substrate solution, xylose isomerase 30 g/L substrate solution. After the mixture was stirred well, the reaction was continued, stirring was continued during the reaction (stirring speed 50 rpm), the reaction temperature was controlled at 40 ° C, and the pH was maintained at 7.0-7.5.
[0044] 待上述反应进行 3h后， 分离出反应液， 并将反应液送入另一反应釜中， 再向反 应液中加入 50mM的烟酰胺、 20mM的 ZnCl 2、 70mM的 Tris-HCl缓冲液以及烟酰 胺磷酸核糖转移酶 50g/L底物溶液， 搅拌均匀后继续反应， 反应过程中持续搅拌 (搅拌速度 50rpm) ， 控制反应温度为 40°C， 维持 pH值为 7.5-8.0， 再反应 3h后即 得烟酰胺单核苷酸粗产品溶液 （含 NMN12.4mM) ， 再经过滤、 纯化、 干燥后即 得烟酰胺单核苷酸成品。 [0044] After the reaction was carried out for 3 hours, the reaction solution was separated, and the reaction solution was sent to another reaction vessel, and 50 mM of nicotinamide, 20 mM of ZnCl 2 , and 70 mM of Tris-HCl buffer were added to the reaction solution. And nicotinamide phosphoribosyltransferase 50g / 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 3h After that, a crude nicotinamide mononucleotide solution (containing NMN12.4 mM) was obtained, which was filtered, purified, and dried to obtain a finished nicotinamide single nucleotide.
[0045] 实施例 4 [0045] Example 4
[0046] 烟酰胺单核苷酸的制备 Preparation of Nicotinamide Mononucleotide
[0047] 向反应釜中加入底物溶液， 含 150mM的烟酰胺、 50mM的 ATP、 50mM的木糖 、 30mM的 MgCl 2、 20mM的 KC1、 10mM的 ZnCl 2以及 lOOmM的 Tris-HCl缓冲液， 调 pH至 8.0-8.5。 然后向底物溶液中加入催化用的各种酶， 各种酶的加入量分别 为： 烟酰胺磷酸核糖转移酶 100g/L底物溶液， 核糖磷酸焦磷酸激酶 100g/L底物溶 液， 核糖 -5-磷酸异构酶 100g/L底物溶液， 核酮糖 - 3-磷酸异构酶 100g/L底物溶液 ， 木酮糖激酶 100g/L底物溶液， 木糖异构酶 100g/L底物溶液。 搅拌均匀后进行反 应， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制反应温度为 50°C， 维持 pH值 为 8.0-8.5。 反应 8h后即得烟酰胺单核苷酸粗产品溶液 （含 NMN22mM) ， 再经过 滤、 纯化、 干燥后即得烟酰胺单核苷酸成品。 [0047] The substrate solution was added to the reaction vessel, containing 150 mM nicotinamide, 50 mM ATP, 50 mM xylose, 30 mM MgCl 2 , 20 mM KC1, 10 mM ZnCl 2 and 100 mM Tris-HCl buffer. pH to 8.0-8.5. Then, 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, ribose- 5-phosphate isomerase 100g/L substrate solution, ribulose-3-phosphate isomerase 100g/L substrate solution, xylulose kinase 100g/L substrate solution, xylose isomerase 100g/L bottom Solution. After the mixture was uniformly stirred, the reaction was continued, stirring was continued during the reaction (stirring speed: 50 rpm), the reaction temperature was controlled at 50 ° C, and the pH was maintained at 8.0 to 8.5. After 8 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN 22 mM) was obtained, which was filtered, purified and dried to obtain a finished nicotinamide single nucleotide.
[0048] 实施例 5  Embodiment 5
[0049] 烟酰胺单核苷酸的制备  Preparation of Nicotinamide Mononucleotide
[0050] 制备固定化酶： 用洗酶缓冲液 （0.02M Tris-HCl/0.001M EDTA， pH7.0溶液） 分别将烟酰胺磷酸核糖转移酶、 核糖磷酸焦磷酸激酶、 核糖 -5-磷酸异构酶、 核 酮糖- 3-磷酸异构酶、 木酮糖激酶以及木糖异构酶稀释至蛋白含量为 5-10mg/ml， 再将酶稀释液与 PB溶液 （2.0mol/L磷酸二氢钾， pH7.5) 等体积混合， 然后加入 酶固定化载体环氧型 LX-3000 (50毫克酶 /克载体） ， 于摇床 （转速 150rpm) 中 2 5°C反应 20小吋。 反应完成后用滤袋过滤， 用洗酶缓冲液清洗 5-6次， 即分别得到 固定化烟酰胺磷酸核糖转移酶、 固定化核糖磷酸焦磷酸激酶、 固定化核糖 -5-磷 酸异构酶、 固定化核酮糖 - 3-磷酸异构酶、 固定化木酮糖激酶以及固定化木糖异 构酶。 Preparation of immobilized enzyme: Nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate, respectively, using a washing enzyme buffer (0.02 M Tris-HCl/0.001 M EDTA, pH 7.0 solution) The enzyme, ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase are diluted to a protein content of 5-10 mg/ml, and the enzyme dilution is combined with PB solution (2.0 mol/L phosphoric acid). Potassium hydrogen, pH 7.5) mixed in equal volumes, then add the enzyme immobilized carrier epoxy type LX-3000 (50 mg enzyme / gram carrier) in a shaker (rotation speed 150 rpm) 2 The reaction was carried out at 5 ° C for 20 hours. 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 immobilized nicotinamide phosphoribosyltransferase, immobilized ribose phosphate pyrophosphate kinase, immobilized ribose-5-phosphate isomerase, Immobilized ribulose-3-phosphate isomerase, immobilized xylulokinase, and immobilized xylose isomerase.
[0051] 向反应釜中加入底物溶液， 含 lOOmM的烟酰胺、 30mM的 ATP、 30mM的木糖 、 12mM的 MgCl 2、 lOmM的 KC1、 lOmM的 ZnCl 2、 以及 lOOmM的 Tris-HCl缓冲液 ， 调 pH至 7.0-7.5。 然后加入催化用的各种酶， 各种酶的加入量为： 固定化烟酰 胺磷酸核糖转移酶 15g/L底物溶液， 固定化核糖磷酸焦磷酸激酶 6g/L底物溶液， 固定化核糖 -5-磷酸异构酶 10g/L底物溶液， 固定化核酮糖- 3-磷酸异构酶 l lg/L底 物溶液， 固定化木酮糖激酶 10g/L底物溶液， 固定化木糖异构酶 10g/L底物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制反应温度 为 37°C， 维持 pH值为 7.0-7.5。 反应 6h后即得烟酰胺单核苷酸粗产品溶液 （含 NM N13.3mM) ， 再经过滤、 纯化、 干燥后即得烟酰胺单核苷酸。 [0051] A substrate solution containing 100 mM of nicotinamide, 30 mM of ATP, 30 mM of xylose, 12 mM of MgCl 2 , 10 mM of KC1, 10 mM of ZnCl 2 , and 100 mM of Tris-HCl buffer was added to the reaction vessel. Adjust the pH to 7.0-7.5. Then, various enzymes for catalysis are added, and the amounts of various enzymes added are: immobilized nicotinamide phosphoribosyltransferase 15 g/L substrate solution, immobilized ribose phosphate pyrophosphate kinase 6 g/L substrate solution, immobilized ribose- 5-phosphate isomerase 10g/L substrate solution, immobilized ribulose-3-phosphate isomerase l lg/L substrate solution, immobilized xylulokinase 10g/L substrate solution, immobilized xylose Isomerase 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 6 h of reaction, a crude nicotinamide mononucleotide solution (containing NM N13.3 mM) was obtained, which was filtered, purified and dried to obtain a nicotinamide mononucleotide.
[0052] 实施例 6  Example 6
[0053] 烟酰胺磷酸核糖转移酶突变体的制备  Preparation of Nicotinamide Phosphoribosyltransferase Mutant
[0054] 本发明提供的方法中用到的人工诱导定点突变的烟酰胺磷酸核糖转移酶的制备 过程大致为： 首先构建含有亲本烟酰胺磷酸核糖转移酶基因的载体质粒， 然后 设定定点突变的位点以及突变后的氨基酸种类， 再合成适当的引物， 以含亲本 烟酰胺磷酸核糖转移酶基因的载体质粒为模板， PCR扩增 DNA片段、 装配所扩 增的 DNA片段以及 PCR扩增全长突变基因。 然后将该全长突变基因克隆到适当 的载体上并转化适当的宿主细胞， 经培养筛选出具有烟酰胺磷酸核糖转移酶活 性的阳性克隆。 最后从阳性克隆中提取质粒 DNA， 进行 DNA序列测定分析， 以 确定引入的突变， 在确定目的片段***到载体上后， 通过 LB+卡那霉素培养基筛 选， 从而获得一系列具有高催化活性的烟酰胺磷酸核糖转移酶突变体。  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.
[0055] 在上述制备方法中， 可采用任何适用的载体， 例如： 可以为原核表达载体， 如 pRSET和 pES21等； 可以为克隆载体， 如 pUC18/19和 pBluscript-SK等。 本发明 优先选用 pRSET-A为载体， 载体的宿主细胞可以是包括大肠杆菌在内的原核细 胞， 也可以是包括酿酒酵母和毕赤巴斯德酵母在内的真核细胞。 [0056] 一、 含有亲本烟酰胺磷酸核糖转移酶基因的载体质粒的构建 In the above preparation method, 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. In the present invention, pRSET-A is preferred as the 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. [0056] 1. Construction of a vector plasmid containing a parent nicotinamide phosphoribosyltransferase gene
[0057] 对基因库公布的来自 Meiothermus ruber DSM 1279的亲本烟酰胺磷酸核糖转移 酶的基因序列 （GenBank登录号： CP001743.1) 进行全序列人工合成 （由商业合 成公司完成） 。 合成的产物经限制性内切酶 Ndel和 BamHI酶切后与经同样限制性 内切酶 Ndel和 BamHI酶切的载体 pRSET-A (源自 Invitrogen, [0057] The gene sequence of the parent nicotinamide phosphoribosyltransferase from Meiusermus ruber DSM 1279 (GenBank accession number: CP001743.1) was subjected to full sequence synthesis (completed by a commercial synthesis company). The synthesized product was digested with restriction endonucleases Ndel and BamHI and digested with the same restriction endonucleases Ndel and BamHI, pRSET-A (from Invitrogen,
USA) 连接， 得质粒 pRSET-nampt。 经 DNA测序， 确定该被克隆的亲本烟酰胺磷 酸核糖转移酶的核苷酸序列如 SEQ ID NO: 1所示， 其氨基酸序列如 SEQ ID NO ： 2所示。 USA) ligation, the plasmid pRSET- n ampt. 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.
[0058] 二、 烟酰胺磷酸核糖转移酶突变体的制备  [0058] Second, the preparation of nicotinamide phosphoribosyltransferase mutant
[0059] PCR扩增反应体系为： 20 mM Tris-HCl (pH 8.8), lO 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聚合酶（Promega, USA) , 20 ng DNA模板 ， 以及 400 nM上游引物和 400 nM下游引物， 用无菌水调反应体积至 50微升。 [0059] 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 μm with sterile water Rise.
[0060] PCR扩增反应条件为： 95°C 3分钟， 35圈循环： 95°C 50秒、 52°C 30秒和 72°C 3 分钟， 最后 72°C 5分钟。  [0060] 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.
[0061] 1、 F180A突变体的制备  1. Preparation of F180A mutant
[0062] 用如下引物对 F180A-F: 5'  [0062] The following primer pairs were used for F180A-F: 5'
GTTCAAACTGCACGACGCGGGTGCTCGTGGTGTTTC 3'和 F180A-R: 5'  GTTCAAACTGCACGACGCGGGTGCTCGTGGTGTTTC 3' and F180A-R: 5'
GAAACACCACGAGCACCCGCGTCGTGCAGTTTGAAC 3'， 以实施例 6第一部 分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应 条件进行高保真 PCR扩增 F180A突变体基因， 经 1%琼脂糖胶电泳分离并用商业 试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第 一部分） ， 得到质粒 pRSET-F180A。 将质粒 pRSET-F180A转化感受态细菌细胞 E. coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟酰 胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-F180A的 DNA， 经 DN A测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相比 ， F180A突变体的氨基酸序列在第 180位点由 Phe (F) 突变为 Ala (A) ， 其氨基 酸序列如 SEQ ID NO: 3所示。 [0063] 2、 F180W突变体的制备 GAAACACCACGAGCACCCGCGTCGTGCAGTTTGAAC 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 the F180A mutant gene, electrophoresed by 1% agarose gel The amplified product was isolated and 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-F180A. The plasmid pRSET-F180A 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-F180A was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the F180A mutant is mutated from Phe (F) to Ala (A) at the 180th position, and the amino acid sequence thereof is as shown in SEQ ID NO: 3, as compared with the parent amino acid sequence shown in SEQ ID NO: 2. . 2. Preparation of F180W mutant
[0064] 用如下引物对 F180W-F: 5'  [0064] Use the following primer pair F180W-F: 5'
GTTCAAACTGCACGACTGGGGTGCTCGTGGTGTTTC 3'和 F180W-R: 5' GAAACACCACGAGCACCCCAGTCGTGCAGTTTGAAC 3'， 以实施例 6第一部 分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应 条件进行高保真 PCR扩增 F180W突变体基因， 经 1%琼脂糖胶电泳分离并用商业 试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第 一部分） ， 得到质粒 pRSET-F180W。 将质粒 pRSET-F180W转化感受态细菌细胞 E. coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟 酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-F180W的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相 比， F180W突变体的氨基酸序列在第 180位点由 Phe (F) 突变为 Trp (w) 。  GTTCAAACTGCACGACTGGGGTGCTCGTGGTGTTTC 3' and F180W-R: 5' GAAACACCACGAGCACCCCAGTCGTGCAGTTTGAAC 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 F180W mutation The 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-F180W. 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.
[0065] 3、 A182Y突变体的制备  3. Preparation of A182Y mutant
[0066] 用如下引物对 A182Y-F: 5'  [0066] The following primer pair A182Y-F: 5'
第一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩 增反应条件进行高保真 PCR扩增 A182Y突变体基因， 经 1%琼脂糖胶电泳分离并 用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实 施例 6第一部分） ， 得到质粒 pRSET-A182Y。 将质粒 pRSET-A182Y转化感受态细 菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出 具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-A182Y的 DN A， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸 序列相比， A182Y突变体的氨基酸序列在第 182位点由 Ala (A) 突变为 Tyr (Y) 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. 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-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.
[0067] 4、 E231A突变体的制备 4. Preparation of E231A mutant
[0068] 用如下弓 I物对 Ε231 A-F: 5' CTATCCCGGCTATGGCGCACTCTACCGTTAC [0068] With the following object Ε 231 A-F: 5' CTATCCCGGCTATGGCGCACTCTACCGTTAC
3'和 Ε231 A-R: 5' GTAACGGTAGAGTGCGCCATAGCCGGGATAG 3'， 以实施例 6 第一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩 增反应条件进行高保真 PCR扩增 E231A突变体基因， 经 1%琼脂糖胶电泳分离并 用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实 施例 6第一部分） ， 得到质粒 pRSET-E231A。 将质粒 pRSET-E231A转化感受态细 菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出 具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-E231A的 DN A， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸 序列相比， E231A突变体的氨基酸序列在第 231位点由 Glu (E) 突变为 Ala (A) 3' and Ε231 AR: 5' GTAACGGTAGAGTGCGCCATAGCCGGGATAG 3', as in Example 6 The plasmid pRSET-nampt constructed in the first part was used as a template, and the E231A 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-E231A. The plasmid pRSET-E231A 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-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 set forth in SEQ ID NO: 2.
[0069] 5、 E231Q突变体的制备 [0069] 5. Preparation of E231Q mutant
[0070] 用如下弓 I物对 E231Q-F: 5' CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3'和 E231Q-R: 5' GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 3'， 以实 施例 6第一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 P CR扩增反应条件进行高保真 PCR扩增 E231Q突变体基因， 经 1%琼脂糖胶电泳分 离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参 考实施例 6第一部分） ， 得到质粒 pRSET-E231Q。 将质粒 pRSET-E231Q转化感受 态细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛 选出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-E231Q 的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨 基酸序列相比， E231Q突变体的氨基酸序列在第 231位点由 Glu (E) 突变为 Gin ( Q) 。  [0070] Using the following pair of E231Q-F: 5' CTCTATCCCGGCTATGCAGCACTCTACCGTTACC 3' and E231Q-R: 5' GGTAACGGTAGAGTGCTGCATAGCCGGGATAGAG 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 P CR amplification reaction conditions 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.
[0071] 6、 D298A突变体的制备  6. Preparation of D298A mutant
[0072] 用如下弓 I物对 D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3'和 D298A-R:  [0072] The following pairs of objects D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3' and D298A-R:
5' GGGTCACCAGACGCCGGACGGATA  5' GGGTCACCAGACGCCGGACGGATA
3'， 以实施例 6第一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应 体系和 PCR扩增反应条件进行高保真 PCR扩增 D298A突变体基因， 经 1%琼脂糖 胶电泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 (具体参考实施例 6第一部分） ， 得到质粒 pRSET-D298A。 将质粒 pRSET-D298A 转化感受态细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉 素） 上筛选出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET -D298A的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示 的亲本氨基酸序列相比， D298A突变体的氨基酸序列在第 298位点由 Asp (D) 突 变为 Ala (A) 。 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 D298A mutant gene, separated by 1% agarose gel electrophoresis 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-D298A. Plasmid pRSET-D298A Transformation of competent bacterial cells E. coli BL21, selection of clones with nicotinamide phosphoribosyltransferase activity on Luria broth (LB) plates (containing 50 mg/L kanamycin), extraction of plasmid pRSET-D298A from clones The DNA was confirmed by DNA sequencing to confirm that the point mutation was introduced. The amino acid sequence of the D298A mutant was mutated from Asp (D) to Ala (A) at position 298 as compared to the parent amino acid sequence set forth in SEQ ID NO: 2.
[0073] 7、 D298N突变体的制备 7. Preparation of D298N mutant
[0074] 用如下弓 I物对 D298N-F: 5' GTTGTTATCCGTCCGAATTCTGGTGACCCGCCG 3'和 D298N-R: 5' CGGCGGGTCACCAGAATTCGGACGGATAACAAC 3'， 以实施 例 6第一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PC R扩增反应条件进行高保真 PCR扩增 D298N突变体基因， 经 1%琼脂糖胶电泳分离 并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考 实施例 6第一部分） ， 得到质粒 pRSET-D298N。 将质粒 pRSET-D298N转化感受态 细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选 出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D298N的 D NA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基 酸序列相比， D298N突变体的氨基酸序列在第 298位点由 Asp (D) 突变为 Asn ( N) 。  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. 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 as shown in SEQ ID NO: 2.
[0075] 8、 D298E突变体的制备  8. Preparation of D298E mutant
[0076] 用如下引物对 D298E-F: 5'  [0076] The following primer pair D298E-F: 5'
GTTGTTATCCGTCCGGAATCTGGTGACCCGCCGTTC 3'和 D298E-R: 5' GAACGGCGGGTCACCAGATTCCGGACGGATAACAAC 3'， 以实施例 6第一咅 分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应 条件进行高保真 PCR扩增 D298E突变体基因， 经 1%琼脂糖胶电泳分离并用商业 试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第 一部分） ， 得到质粒 pRSET-D298E。 将质粒 pRSET-D298E转化感受态细菌细胞 E . coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟酰 胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D298E的 DNA， 经 DN A测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相比 ， D298E突变体的氨基酸序列在第 298位点由 Asp (D) 突变为 Glu (E GTTGTTATCCGTCCGGAATCTGGTGACCCGCCGTTC 3' and 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. Compared to the parent amino acid sequence as shown in SEQ ID NO: , the amino acid sequence of the D298E mutant was mutated from Asp (D) to Glu at position 298 (E
[0077] 9、 D338N突变体的制备 9. Preparation of D338N mutant
[0078] 用如下引物对 D338N-F: 5'[0078] The following primer pairs were used for D338N-F: 5'
Figure imgf000014_0001
Figure imgf000014_0001
GAGTCAGCGTTAACACCATTACCCTGGATAACACGAAC 3'， 以实施例 6第一 部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反 应条件进行高保真 PCR扩增 D338N突变体基因， 经 1%琼脂糖胶电泳分离并用商 业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6 第一部分） ， 得到质粒 pRSET-D338N。 将质粒 pRSET-D338N转化感受态细菌细 胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有 烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D338N的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列 相比， D338N突变体的氨基酸序列在第 338位点由 Asp (D) 突变为 Asn (N) 。  GAGTCAGCGTTAACACCATTACCCTGGATAACACGAAC 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 D338N mutant gene, electrophoresed by 1% agarose gel The amplified product was isolated and 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 the plasmid pRSET-D338N. The plasmid pRSET-D338N 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-D338N was extracted, and the point mutation introduced was confirmed by DNA sequencing. The amino acid sequence of the D338N mutant was mutated from Asp (D) to Asn (N) at position 338 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
[0079] 10、 D338E突变体的制备 10. Preparation of D338E mutant
[0080] 用如下弓 I物对 D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC [0080] With the following pair of objects D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC
3'和 D338E-R: 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3'， 以实施例 6第 一部分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增 反应条件进行高保真 PCR扩增 D338E突变体基因， 经 1%琼脂糖胶电泳分离并用 商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施 例 6第一部分） ， 得到质粒 pRSET-D338E。 将质粒 pRSET-D338E转化感受态细菌 细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具 有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D338E的 DNA ， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID N0: 2所示的亲本氨基酸序 列相比， D338E突变体的氨基酸序列在第 338位点由 Asp (D) 突变为 Glu (E) 。 3' and 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.
[0081] 11、 D377A突变体的制备  11. Preparation of D377A mutant
[0082] 用如下弓 I物对 D377A-F: 5' CACCCGCACCGTGCGACCCAGAAATTC  [0082] The following pairs of objects D377A-F: 5' CACCCGCACCGTGCGACCCAGAAATTC
3'和 D377A-R: 5' GAATTTCTGGGTCGCACGGTGCGGGTG 3'， 以实施例 6第一咅 分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应 条件进行高保真 PCR扩增 D377A突变体基因， 经 1%琼脂糖胶电泳分离并用商业 试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第 一部分） ， 得到质粒 pRSET-D377A。 将质粒 pRSET-D377A转化感受态细菌细胞 E. coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟 酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D377A的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相 比， D377A突变体的氨基酸序列在第 377位点由 Asp (D) 突变为 Ala (A) 。 3' and 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 The high-fidelity PCR amplification of the D377A mutant gene was performed 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-D377A was obtained. The plasmid pRSET-D377A 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-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.
[0083] 12、 D377N突变体的制备  12. Preparation of D377N Mutant
[0084] 用如下引物对 D377N-F: 5'  [0084] The following primer pairs were used for D377N-F: 5'
GAGCGAATTTCTGGGTATTACGGTGCGGGTGTTGC 3'， 以实施例 6第一咅吩 构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应条 件进行高保真 PCR扩增 D377N突变体基因， 经 1%琼脂糖胶电泳分离并用商业试 剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第一 部分） ， 得到质粒 pRSET-D377N。 将质粒 pRSET-D377N转化感受态细菌细胞 E. coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟酰 胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D377N的 DNA， 经 DN A测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相比 ， D377N突变体的氨基酸序列在第 377位点由 Asp (D) 突变为 Asn (N) 。 GAGCGAATTTCTGGGTATTACGGTGCGGGTGTTGC 3', using the plasmid pRSET-nampt constructed in the first porphin of Example 6 as a template, using the above PCR amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of the D377N mutant gene, 1% agarose The amplified product was separated by gel electrophoresis and 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-D377N. The plasmid pRSET-D377N 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-D377N was extracted, and the point mutation introduced was confirmed by DN A sequencing. The amino acid sequence of the D377N mutant was mutated from Asp (D) to Asn (N) at position 377 as compared to the parent amino acid sequence as shown in SEQ ID NO: 2.
[0085] 13、 D377E突变体的制备 13. Preparation of D377E Mutant
[0086] 用如下弓 I物对 D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG  [0086] With the following pair of objects D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG
3'和 D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3'， 以实施例 6第一部 分构建的质粒 pRSET-nampt为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应 条件进行高保真 PCR扩增 D377E突变体基因， 经 1%琼脂糖胶电泳分离并用商业 试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具体参考实施例 6第 一部分） ， 得到质粒 pRSET-D377E。 将质粒 pRSET-D377E转化感受态细菌细胞 E . coli BL21 , 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟酰 胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-D377E的 DNA， 经 DN A测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序列相比 ， D377E突变体的氨基酸序列在第 377位点由 Asp (D) 突变为 Glu (E) 。 3' and 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. coli BL21, and a clone having nicotinamide phosphoribosyltransferase activity was screened on Luria broth (LB) plate (containing 50 mg/L kanamycin) from the clone. DNA extracted from plasmid pRSET-D377E, via DN A sequencing confirmed that the introduced point mutation was correct. The amino acid sequence of the D377E mutant was mutated from Asp (D) to Glu (E) at position 377 as compared to the parent amino acid sequence set forth in SEQ ID NO: 2.
[0087] 14、 E231Q/D338E突变体的制备 [0087] 14. Preparation of E231Q/D338E Mutant
[0088] 用如下引物对 D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC [0088] The following primer pairs were used for D338E-F: 5' GTTATCCAGGGTGAAGGTGTTAACGCTGAC
3'和 D338E-R: 5' GTCAGCGTTAACACCTTCACCCTGGATAAC 3', 以实施例 6 第二部分的第 5小节构建的质粒 pRSET-E231Q为模板， 利用上述 PCR扩增反应体 系和 PCR扩增反应条件进行高保真 PCR扩增 E231Q/D338E突变体基因， 经 1%琼 脂糖胶电泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A 连接 （具体参考实施例 6第一部分） ， 得到质粒 pRSET-21。 将质粒 pRSET-21转 化感受态细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素 ) 上筛选出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-2 1的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本 氨基酸序列相比， E231Q/D338E突变体的氨基酸序列在第 231位点由 Glu (E) 突 变为 Gin (Q) 、 在第 338位点由 Asp (D) 突变为 Glu (E) 。 3' and 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、 E231Q/D377E突变体的制备 [0089] 15. Preparation of E231Q/D377E mutant
[0090] 用如下引物对 D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG  [0090] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG
3'和 D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', 以实施例 6第二部 分的第 5小节构建的质粒 pRSET-E231Q为模板， 利用上述 PCR扩增反应体系和 PC R扩增反应条件进行高保真 PCR扩增 E231Q/D377E突变体基因， 经 1%琼脂糖胶电 泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具 体参考实施例 6第一部分） ， 得到质粒 pRSET-22。 将质粒 pRSET-22转化感受态 细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选 出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-22的 DNA ， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序 列相比， E231Q/D377E突变体的氨基酸序列在第 231位点由 Glu (E) 突变为 Gin (Q) 、 在第 377位点由 Asp (D) 突变为 Glu (E) 。  3' and 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).
[0091] 16、 D338E/D377E突变体的制备 16. Preparation of D338E/D377E Mutant
[0092] 用如下引物对 D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG 3'和 D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', 以实施例 6第二部 分的第 10小节构建的质粒 pRSET-D338E为模板， 利用上述 PCR扩增反应体系和 Ρ CR扩增反应条件进行高保真 PCR扩增 D338E/D377E突变体基因， 经 1%琼脂糖胶 电泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （ 具体参考实施例 6第一部分） ， 得到质粒 pRSET-23。 将质粒 pRSET-23转化感受 态细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛 选出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-23的 DN A， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸 序列相比， D338E/D377E突变体的氨基酸序列在第 338位点由 Asp (D) 突变为 G1 u (E) 、 在第 377位点由 Asp (D) 突变为 Glu (E) 。 [0092] The following primer pair D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG 3' and 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 ΡCR 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. 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. 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).
[0093] 17、 E231Q/D338E/D377E突变体的制备 17. Preparation of E231Q/D338E/D377E Mutant
[0094] 用如下引物对 D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG  [0094] The following primer pairs were used for D377E-F: 5' CCCGCACCGTGAAACCCAGAAATTCG
3'和 D377E-R: 5' CGAATTTCTGGGTTTCACGGTGCGGG 3', 以实施例 6第二部 分的第 14小节构建的质粒 pRSET-21为模板， 禾 上述 PCR扩增反应体系和 PCR 扩增反应条件进行高保真 PCR扩增 E231Q/D338E/D377E突变体基因， 经 1%琼脂 糖胶电泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连 接 （具体参考实施例 6第一部分） ， 得到质粒 pRSET-31。 将质粒 pRSET-31转化 感受态细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-31 的 DNA， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨 基酸序列相比， E231Q/D338E/D377E突变体的氨基酸序列在第 231位点由 Glu (E ) 突变为 Gin (Q) 、 在第 338位点由 Asp (D) 突变为 Glu (E) 、 在第 377位点由 Asp (D) 突变为 Glu (E) 。  3' and 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.
[0095] 18、 E231Q/D298A/D338E/D377E突变体的制备 18. Preparation of E231Q/D298A/D338E/D377E Mutants
[0096] 用如下引物对 D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3'和 D298A-R ： 5' GGGTCACCAGACGCCGGACGGATA 3', 以实施例 6第二部分的第 17小节 构建的质粒 pRSET-31为模板， 利用上述 PCR扩增反应体系和 PCR扩增反应条件 进行高保真 PCR扩增 E231Q/D298A/D338E/D377E突变体基因， 经 1%琼脂糖胶电 泳分离并用商业试剂盒回收扩增产物， 再将扩增产物用载体 pRSET-A连接 （具 体参考实施例 6第一部分） ， 得到质粒 pRSET-41。 将质粒 pRSET-41转化感受态 细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上筛选 出具有烟酰胺磷酸核糖转移酶活性的克隆， 从克隆中提取质粒 pRSET-41的 DNA ， 经 DNA测序确定引入的点突变无误。 与如 SEQ ID NO: 2所示的亲本氨基酸序 列相比， E231Q/D298A/D338E/D377E突变体的氨基酸序列在第 231位点由 Glu (E ) 突变为 Gin (Q) 、 在第 298位点由 Asp (D) 突变为 Ala (A) 、 在第 338位点由 Asp (D) 突变为 Glu (E) 、 在第 377位点由 Asp (D) 突变为 Glu (E) 。 Using the following primer pair D298A-F: 5' TATCCGTCCGGCGTCTGGTGACCC 3' and D298A-R: 5' GGGTCACCAGACGCCGGACGGATA 3', 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 Amplification reaction system and PCR amplification reaction conditions for high-fidelity PCR amplification of E231Q/D298A/D338E/D377E mutant gene, 1% agarose gel The amplified product was isolated by swimming and recovered with 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 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. 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.
[0097] 三、 酶的提取 [0097] Third, the extraction of enzymes
[0098] 将含亲本烟酰胺磷酸核糖转移酶基因的质粒 pRSET-nampt以及含烟酰胺磷酸核 糖转移酶突变体基因的质粒 pRSET-F180A、 pRSET-F180W、 pRSET-A182Y、 pR SET-E231A、 pRSET-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以及 pRSET-41分别转化感受态 细菌细胞 E. coli BL21， 在 Luria broth (LB) 平板 （含 50 mg/L卡那霉素） 上 37°C 培养 24小吋。 接种单个克隆于 50毫升 LB液体培养基 （含 50  The plasmid pRSET-nampt containing the parent nicotinamide phosphoribosyltransferase gene and the plasmid pRSET-F180A, pRSET-F180W, pRSET-A182Y, pR SET-E231A, pRSET- containing the nicotinamide phosphoribosyltransferase mutant gene. 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
mg/L卡那霉素） 中于 30°C培养 16-20小吋。 离心收集菌体， 称量等同量菌体按照 1 :4比例悬浮破菌液 （pH  In mg/L kanamycin), culture at 30 ° C for 16-20 hours. The cells were collected by centrifugation, and the equivalent amount of bacteria was weighed and suspended in a ratio of 1:4 (pH).
7.5) 中。 然后用超声波裂解细菌细胞。 离心 （4-10°C， 12000rpm, 10分钟） 并 收集上清液， 即分别得到亲本烟酰胺磷酸核糖转移酶以及一系列烟酰胺磷酸核 糖转移酶突变体的上清蛋白溶液， 可用于酶活性的测定以及烟酰胺单核苷酸的 的生物催化制备。  7.5) Medium. The bacterial cells are then lysed with ultrasound. Centrifuge (4-10 ° C, 12000 rpm, 10 minutes) and collect the supernatant to obtain the parent nicotinamide phosphoribosyltransferase and a series of supernatant proteins of nicotinamide phosphoribosyltransferase mutants, which can be used for enzyme activity. Determination of the biocatalytic preparation of nicotinamide mononucleotides.
[0099] 四、 酶活性的测定 [0099] Fourth, the determination of enzyme activity
[0100] 配制底物溶液： 含 60mM的烟酰胺、 25mM的 PRPP、 18mM的 MgCl 2 Formulation of substrate solution: 60 mM nicotinamide, 25 mM PRPP, 18 mM MgCl 2
、 15mM的 KC1和 lOOmM的 Tris buffer缓冲液， 调 pH至 7.5。 分别取底物溶液 900微 升各 19份， 然后分别加入 100微升等浓度的的实施例 6第三部分得到的亲本烟酰 胺磷酸核糖转移酶以及各烟酰胺磷酸核糖转移酶突变体的上清蛋白溶液， 于 37°C 进行 10分钟反应， 加入 10( L25%三氯乙酸终止反应。 通过高效液相色谱仪 （HP LC) 测定反应液中烟酰胺单核苷酸 （NMN) 的含量， 并计算每种酶的比酶活， 以亲本烟酰胺磷酸核糖转移酶的比酶活为参比 100， 亲本及各突变体的相对比活 性如表 1所示。 , 15 mM KC1 and 100 mM Tris buffer buffer, adjusted to pH 7.5. Take 900 μl of each substrate solution, respectively, and then add 100 μl of the same concentration of the parent nicotinamide phosphoribosyltransferase obtained in the third part of Example 6 and the supernatant of each nicotinamide phosphoribosyltransferase mutant. The protein solution was reacted at 37 ° C for 10 minutes, and 10 (L25% trichloroacetic acid was added to terminate the reaction. By high performance liquid chromatography (HP) LC) Determine the content of nicotinamide mononucleotide (NMN) in the reaction solution, and calculate the specific enzyme activity of each enzyme. The specific enzyme activity of the parent nicotinamide phosphoribosyltransferase is reference 100, the parent and each mutant. The relative specific activities are shown in Table 1.
表 1烟酰胺磷酸核糖转移酶的酶活  Table 1 Enzyme activity of nicotinamide phosphoribosyltransferase
[表 1] [Table 1]
Figure imgf000019_0001
[0102] 五、 烟酰胺单核苷酸的制备
Figure imgf000019_0001
V. Preparation of Nicotinamide Mononucleotide
[0103] 向反应釜中加入底物溶液， 含 30mM的烟酰胺、 20mM的 ATP、 30mM的木糖、 12mM的 MgCl 2、 lOmM的 KC1、 lOmM的 ZnCl 2、 以及 lOOmM的 Tris-HCl缓冲液， 调 pH至 7.0-7.5。 然后加入催化用的各种酶， 各种酶的加入量为： 实施例 6第三部 分得到的烟酰胺磷酸核糖转移酶突变体 （F180A) 的上清蛋白溶液 10ml /L底物 溶液， 核糖磷酸焦磷酸激酶 6g/L底物溶液， 核糖 -5-磷酸异构酶 10g/L底物溶液， 核酮糖-[0103] A substrate solution containing 30 mM nicotinamide, 20 mM ATP, 30 mM xylose, 12 mM MgCl 2 , 10 mM KC1, 10 mM ZnCl 2 , and 100 mM Tris-HCl buffer was added to the reaction vessel. Adjust the pH to 7.0-7.5. Then, various enzymes for catalysis were added, 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 10 ml / L substrate solution, ribose phosphate Pyrophosphokinase 6g/L substrate solution, ribose-5-phosphate isomerase 10g/L substrate solution, ribulose-
3-磷酸异构酶 l lg/L底物溶液， 木酮糖激酶 10g/L底物溶液， 木糖异构酶 10g/L底 物溶液。 搅拌均匀后进行反应， 反应过程中持续搅拌 （搅拌速度 50rpm) ， 控制 反应温度为 37°C， 维持 pH值为 7.0-7.5。 反应 6h后即得烟酰胺单核苷酸粗产品溶 液 （含 NMNlOmM) ， 再经过滤、 纯化、 干燥后即得烟酰胺单核苷酸。 3-phosphate isomerase l lg/L substrate solution, xylulose kinase 10 g/L substrate solution, xylose isomerase 10 g/L substrate solution. After the mixture was stirred well, the reaction was continued, 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 6 hours of reaction, a crude nicotinamide mononucleotide solution (containing NMN10 mM) was obtained, which was filtered, purified and dried to obtain a nicotinamide mononucleotide.

Claims

权利要求书 Claim
一种制备烟酰胺单核苷酸的方法， 其特征在于： 以烟酰胺、 ATP和木 糖为原料， 在烟酰胺磷酸核糖转移酶、 核糖磷酸焦磷酸激酶、 核糖 -5 -磷酸异构酶、 核酮糖 - 3-磷酸异构酶、 木酮糖激酶以及木糖异构酶的 催化作用下发生反应， 制得烟酰胺单核苷酸。 A method for preparing a nicotinamide mononucleotide, which comprises: nicotinamide, ATP and xylose as raw materials, nicotinamide phosphoribosyltransferase, ribose phosphate pyrophosphate kinase, ribose-5-phosphate isomerase, The reaction of ribulose-3-phosphate isomerase, xylulokinase and xylose isomerase catalyzes to produce a nicotinamide mononucleotide.
根据权利要求 1所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述反应在温度为 30-50°C， pH值为 6.5-8.5的条件下进行。 The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out at a temperature of 30 to 50 ° C and a pH of 6.5 to 8.5.
根据权利要求 1所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述反应在 Mg 2+、 K +和 Zn 2+存在的条件下进行。 The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out in the presence of Mg 2+ , K + and Zn 2+ .
根据权利要求 1所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述反应在 Tris-HCl缓冲液中进行。 The method for producing a nicotinamide mononucleotide according to claim 1, wherein the reaction is carried out in a Tris-HCl buffer.
根据权利要求 1所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述烟酰胺的浓度为 l-150mM， 所述 ATP的浓度为 l-50mM， 所述木糖 的浓度为 l-50mM。 The method for preparing a nicotinamide mononucleotide according to claim 1, wherein: the concentration of the nicotinamide is 1-150 mM, the concentration of the ATP is 1-50 mM, and the concentration of the xylose is l. -50 mM.
根据权利要求 1所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述原料中烟酰胺、 ATP、 木糖的摩尔比为 1-4:1:1-4。 The method for producing a nicotinamide mononucleotide according to claim 1, wherein the molar ratio of nicotinamide, ATP, and xylose in the raw material is 1-4:1:1-4.
根据权利要求 6所述的制备烟酰胺单核苷酸的方法， 其特征在于： 所 述原料中烟酰胺、 ATP、 木糖的摩尔比为 1.5:1:1.5。 The method for producing a nicotinamide mononucleotide according to claim 6, wherein the molar ratio of nicotinamide, ATP, and xylose in the raw material is 1.5:1:1.5.
根据权利要求 1至 7任一项所述的制备烟酰胺单核苷酸的方法， 其特征 在于所述烟酰胺磷酸核糖转移酶为如下 ) 或 （b) 的蛋白质：The method for producing a nicotinamide mononucleotide according to any one of claims 1 to 7, wherein the nicotinamide phosphoribosyltransferase is a protein of the following: or (b):
(a) 其氨基酸序列如 SEQ ID NO: 3所示的蛋白质， (a) a protein having the amino acid sequence of SEQ ID NO: 3,
(b) 在 （a) 限定的氨基酸序列中经过取代、 缺失或添加一个或几个 氨基酸并且以烟酰胺和 PRPP为底物具有比氨基酸序列如 SEQ ID NO (b) Substituting, deleting or adding one or several amino acids in the (a) defined amino acid sequence and having a specific amino acid sequence such as SEQ ID NO with nicotinamide and PRPP as substrates
： 2所示的亲本高的烟酰胺磷酸核糖转移酶催化活性的由 （a) 衍生的 蛋白质。 : 2 The parental high nicotinamide phosphoribosyltransferase catalytically active protein derived from (a).
根据权利要求 8所述的制备烟酰胺单核苷酸的方法， 其特征在于所述 烟酰胺磷酸核糖转移酶与如 SEQ ID NO: 2所示的氨基酸序列相比在 选自至少一个下述位点处具有至少一个突变： 第 180位、 第 182位、 第 231位、 第 298位、 第 338位以及第 377位。 The method for producing a nicotinamide mononucleotide according to claim 8, wherein the nicotinamide phosphoribosyltransferase is selected from at least one of the following positions as compared with the amino acid sequence shown in SEQ ID NO: There are at least one mutation at the point: 180th, 182th, first 231, 298, 338 and 377.
[权利要求 10] 根据权利要求 9所述的制备烟酰胺单核苷酸的方法， 其特征在于所述 烟酰胺磷酸核糖转移酶具有至少一个下述突变： F180A、 F180W、 Al 82Y、 Ε231Α、 E231Q、 D298A、 D298N、 D298E、 D338N、 D338E、 D377A、 D377N以及 D377E。 [Claim 10] The method for producing a nicotinamide mononucleotide according to claim 9, wherein the nicotinamide phosphoribosyltransferase has at least one of the following mutations: F180A, F180W, Al 82Y, Ε231Α, E231Q , D298A, D298N, D298E, D338N, D338E, D377A, D377N and D377E.
PCT/CN2016/092457 2016-07-30 2016-07-30 Method for preparing nicotinamide mononucleotide WO2018023207A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201680003960.0A CN107889504B (en) 2016-07-30 2016-07-30 Method for preparing nicotinamide mononucleotide
PCT/CN2016/092457 WO2018023207A1 (en) 2016-07-30 2016-07-30 Method for preparing nicotinamide mononucleotide

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2016/092457 WO2018023207A1 (en) 2016-07-30 2016-07-30 Method for preparing nicotinamide mononucleotide

Publications (1)

Publication Number Publication Date
WO2018023207A1 true WO2018023207A1 (en) 2018-02-08

Family

ID=61072141

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/092457 WO2018023207A1 (en) 2016-07-30 2016-07-30 Method for preparing nicotinamide mononucleotide

Country Status (2)

Country Link
CN (1) CN107889504B (en)
WO (1) WO2018023207A1 (en)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
CN110483601A (en) * 2019-08-12 2019-11-22 上海龙翔生物医药开发有限公司 Prepare β-niacinamide usp mononucleotide method and its application
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
US11180521B2 (en) 2018-01-30 2021-11-23 Metro International Biotech, Llc Nicotinamide riboside analogs, pharmaceutical compositions, and uses thereof
CN113811770A (en) * 2019-05-13 2021-12-17 豪夫迈·罗氏有限公司 Interference-suppressing pharmacokinetic immunoassay
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 (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112167638A (en) * 2020-06-29 2021-01-05 武汉林宝莱生物科技有限公司 Plant antioxidant formula and preparation method
CN114164190B (en) * 2021-10-12 2023-11-21 南宁邦尔克生物技术有限责任公司 Fusion enzyme for producing nicotinamide mononucleotide and application thereof
CN114317515B (en) * 2021-12-31 2024-04-09 内蒙古金达威药业有限公司 Method for preparing beta-nicotinamide mononucleotide by immobilized whole-cell catalysis with modified diatomite as carrier

Citations (3)

* 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
CN102985540A (en) * 2010-06-29 2013-03-20 纳幕尔杜邦公司 Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity
WO2015069860A1 (en) * 2013-11-06 2015-05-14 President And Fellows Of Harvard College Biological production of nad precursors and analogs

Patent Citations (3)

* 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
CN102985540A (en) * 2010-06-29 2013-03-20 纳幕尔杜邦公司 Improved xylose utilization in recombinant zymomonas having increased ribose-5-phosphate activity
WO2015069860A1 (en) * 2013-11-06 2015-05-14 President And Fellows Of Harvard College Biological production of nad precursors and analogs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
"EC 2.7.6.1", IUBMB ENZYME NOMENCLATURE DATABASE, 31 December 1961 (1961-12-31), XP055460566 *
"EC 5.1.3.1", IUBMB ENZYME NOMENCLATURE DATABASE, 31 December 1989 (1989-12-31), XP055460563 *
DATABASE NCBI [O] 11 December 2013 (2013-12-11), "CP001743.1", XP055460569, Database accession no. CP001743.1 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11464796B2 (en) 2015-08-05 2022-10-11 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US11878027B2 (en) 2015-08-05 2024-01-23 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10548913B2 (en) 2015-08-05 2020-02-04 Metro International Biotech, Llc Nicotinamide mononucleotide derivatives and their uses
US10392416B2 (en) 2015-10-02 2019-08-27 Metro International Biotech, Llc Crystal forms of beta-nicotinamide mononucleotide
US11059847B2 (en) 2015-10-02 2021-07-13 Metro International Biotech, Llc Crystal forms of β-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
CN113811770A (en) * 2019-05-13 2021-12-17 豪夫迈·罗氏有限公司 Interference-suppressing pharmacokinetic immunoassay
CN110483601A (en) * 2019-08-12 2019-11-22 上海龙翔生物医药开发有限公司 Prepare β-niacinamide usp mononucleotide method and its application
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
US11959116B2 (en) 2021-10-27 2024-04-16 Asahi Kasei Pharma Corporation Method for producing nicotinamide mononucleotide

Also Published As

Publication number Publication date
CN107889504A (en) 2018-04-06
CN107889504B (en) 2021-07-27

Similar Documents

Publication Publication Date Title
WO2017185549A1 (en) Method for preparing nicotinamide mononucleotide 2
WO2018023207A1 (en) Method for preparing nicotinamide mononucleotide
WO2018023209A1 (en) Method for preparing nicotinamide mononucleotide
WO2018023208A1 (en) Method for preparing nicotinamide mononucleotide
WO2018023210A1 (en) Method for preparing nicotinamide mononucleotide
JP6712356B2 (en) Nicotinamide phosphoribosyl transferase mutant and its application
CN111718915B (en) Nicotinamide phosphoribosyl transferase mutant, recombinant expression vector and recombinant bacterium containing mutant and application
CN109609474B (en) Amino acid dehydrogenase mutant and application thereof in synthesis of L-glufosinate-ammonium
CN109750009B (en) Glufosinate-ammonium dehydrogenase mutant and application thereof
US10519429B2 (en) Nicotinamide phosphoribosyltransferase (NAMPT) mutant and use thereof
CN110791484B (en) Glufosinate-ammonium dehydrogenase mutant and application thereof in production of L-glufosinate-ammonium
CN113832125B (en) Nicotinamide ribokinase mutant and encoding gene and application thereof
CN113151198B (en) Gamma-glutamine synthetase mutant, coding gene, amino acid sequence and application thereof
CN109609478B (en) α -transaminase and mutant thereof and application thereof in asymmetric synthesis of L-glufosinate-ammonium
JP2021530216A (en) Manipulated pantothenate kinase variant enzyme
JP2020536508A (en) Transaminase mutants and their use
CN113151199B (en) Gamma-glutamine synthetase mutant with thermal stability, coding gene, amino acid sequence and application thereof
CN112980906A (en) Enzyme composition for preparing beta-nicotinamide mononucleotide and application thereof
CN116200353A (en) Carbonyl reductase mutant, recombinant bacterium and application thereof
CN111057697A (en) High-temperature-resistant TIM barrel protein mutant and application thereof
CN110452899B (en) Glucose isomerase, mutant and application of mutant in preparation of D-fructose
JP2002045185A (en) F0f1-atpase and dna encoding the enzyme
CN117143844A (en) NADH pyrophosphatase suitable for alkaline pH and application thereof
CN116904421A (en) NADH pyrophosphatase mutant with enhanced heat resistance and application thereof
CN116855561A (en) Process for preparing reduced nicotinamide mononucleotide under high-temperature and high-pH conditions

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16910805

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16910805

Country of ref document: EP

Kind code of ref document: A1