WO2022217827A1 - 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 - Google Patents
一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 Download PDFInfo
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- WO2022217827A1 WO2022217827A1 PCT/CN2021/117960 CN2021117960W WO2022217827A1 WO 2022217827 A1 WO2022217827 A1 WO 2022217827A1 CN 2021117960 W CN2021117960 W CN 2021117960W WO 2022217827 A1 WO2022217827 A1 WO 2022217827A1
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- enzyme
- nicotinamide
- nmn
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- 108090000790 Enzymes Proteins 0.000 title claims abstract description 246
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- 239000000203 mixture Substances 0.000 title claims abstract description 23
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 claims abstract description 68
- 238000000034 method Methods 0.000 claims abstract description 46
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims abstract description 40
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- 238000006555 catalytic reaction Methods 0.000 claims abstract description 13
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- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 18
- 229910019142 PO4 Inorganic materials 0.000 claims description 17
- 239000010452 phosphate Substances 0.000 claims description 17
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- 241000588724 Escherichia coli Species 0.000 claims description 9
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- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 claims description 3
- 241000193755 Bacillus cereus Species 0.000 claims description 3
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- 238000006243 chemical reaction Methods 0.000 abstract description 93
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- JLEBZPBDRKPWTD-TURQNECASA-O N-ribosylnicotinamide Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](CO)O2)O)=C1 JLEBZPBDRKPWTD-TURQNECASA-O 0.000 abstract description 11
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- 230000002427 irreversible effect Effects 0.000 abstract description 4
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- OOXNYFKPOPJIOT-UHFFFAOYSA-N 5-(3-bromophenyl)-7-(6-morpholin-4-ylpyridin-3-yl)pyrido[2,3-d]pyrimidin-4-amine;dihydrochloride Chemical compound Cl.Cl.C=12C(N)=NC=NC2=NC(C=2C=NC(=CC=2)N2CCOCC2)=CC=1C1=CC=CC(Br)=C1 OOXNYFKPOPJIOT-UHFFFAOYSA-N 0.000 description 1
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- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 1
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- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- JESHZQPNPCJVNG-UHFFFAOYSA-L magnesium;sulfite Chemical compound [Mg+2].[O-]S([O-])=O JESHZQPNPCJVNG-UHFFFAOYSA-L 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
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- 235000007079 manganese sulphate Nutrition 0.000 description 1
- MIVBAHRSNUNMPP-UHFFFAOYSA-N manganese(2+);dinitrate Chemical compound [Mn+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O MIVBAHRSNUNMPP-UHFFFAOYSA-N 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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Classifications
-
- 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
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1077—Pentosyltransferases (2.4.2)
-
- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02001—Purine-nucleoside phosphorylase (2.4.2.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01022—Ribosylnicotinamide kinase (2.7.1.22)
Definitions
- the present invention relates to the field of biotechnology, in particular to an enzyme composition for preparing beta-nicotinamide mononucleotide and its application.
- NMN ⁇ -Nicotinamide mononucleotide
- NAD+ nicotinamide adenine dinucleotide
- NAD+ Because the molecular weight of NAD+ is too large, it cannot be taken into cells orally, and it mainly depends on the synthesis of cells in vivo, and the synthesis amount is very low.
- NMN a small molecule precursor of NAD+
- eating ⁇ -NMN can effectively increase the content of NAD+ in the body, and significantly inhibit the metabolism caused by aging, making ⁇ -NMN a "magic medicine”.
- nicotinamide mononucleotide has many health care applications such as delaying aging, treating Parkinson's and other geriatric diseases, regulating insulin secretion, and affecting mRNA expression.
- the main methods for synthesizing NMN include chemical synthesis and biocatalysis.
- the chemical method has high cost and causes serious environmental pollution, and has been gradually replaced by the biological catalysis method.
- biological enzymatic catalyzed production of NMN is more efficient, lower cost, energy saving and environmentally friendly.
- biocatalytic methods for the production of NMN There are three biocatalytic methods for the production of NMN. The first one uses nicotinamide ribose as a raw material and generates NMN under the supply of ATP by nicotinamide ribokinase (Ribosylnicotinamide kinase, EC 2.7.1.22).
- the second is based on nicotinamide, ribose and ATP as substrates, through D-ribokinase (Ribokinase, EC2.7.1.15), nucleic acid phosphate pyrophosphokinase (ribose phosphate pyrophosphokinase, EC2.7.6.1), and nicotinamide Ribose phosphoribosyltransferase (Nicotinamide phosphoribosyltransferase, EC.2.4.2.12) catalyzes the reaction to generate NMN.
- D-ribokinase Ribokinase, EC2.7.1.15
- nucleic acid phosphate pyrophosphokinase ribose phosphate pyrophosphokinase, EC2.7.6.1
- nicotinamide Ribose phosphoribosyltransferase Nicotinamide phosphoribosyltransferas
- the third is adenosine or AMP, ATP, nicotinamide as raw materials, through adenosine kinase (EC 2.7.1.20) (this enzyme is not required when AMP is used as raw material), adenine phosphoribosyltransferase (EC 2.4.2.7 ), nicotinamide phosphoribosyltransferase (Nicotinamide phosphoribosyltransferase, EC.2.4.2.12) catalyzes the generation of NMN.
- the above-mentioned first method directly uses nicotinamide ribose as a raw material, and the substrate conversion rate is high, but the nicotinamide ribose is expensive and has no cost advantage.
- the second and third methods both use PRPP and nicotinamide to prepare NMN through nicotinamide phosphoribosyltransferase (EC.2.4.2.12). Because nicotinamide phosphoribosyltransferase catalyzes a reversible reaction, NMN can also be hydrolyzed while synthesizing NMN, and the reaction conversion rate is low. At the same time, the intermediate PRPP compound is unstable, which is not conducive to the reaction.
- the multi-step reaction of the above two methods requires ATP to participate in the reaction, and the entire process route needs to consume a large amount of ATP, which leads to the still high production cost of the biocatalysis method. It is necessary to develop a more efficient and low-cost new method for biocatalytic preparation of NMN.
- the purpose of the present invention is to provide an enzyme composition for preparing ⁇ -nicotinamide mononucleotide (NMN), so that it can synthesize ⁇ -nicotinamide mononucleotide using adenosine and nicotinamide as raw materials
- NMN ⁇ -nicotinamide mononucleotide
- 1 molecule of NMN is generated, only 1 molecule of ATP needs to be consumed, and the reaction of synthesizing NMN in the last step of the reaction is irreversible, which can greatly improve the substrate conversion rate and reduce production costs;
- Another object of the present invention is to provide a method for synthesizing NMN using the above-mentioned enzyme composition and to provide related applications of the above-mentioned enzyme composition in synthesizing NMN.
- the present invention provides the following technical solutions:
- An enzyme composition for preparing beta-nicotinamide mononucleotide comprising purine-nucleoside phosphorylase (purine-nucleoside phosphorylase, abbreviated as PNP enzyme) with EC numbering EC 2.4.2.1 and EC numbering EC 2.7. 1.22 nicotinamide ribokinase (ribosylnicotinamide kinase, abbreviated as NRK enzyme).
- the present invention provides another technique for synthesizing ⁇ -nicotinamide mononucleotide with adenosine and nicotinamide by utilizing the enzymatic reaction technology, and the reaction process is as follows:
- Adenosine and phosphate first synthesize D-ribose-1-phosphate under the action of PNP enzyme, and then continue to synthesize nicotinamide ribose with nicotinamide under the catalysis of this enzyme, and then consume a molecule of ATP to irreversibly react under the catalysis of NRK enzyme to generate NMN.
- the PNP enzyme contains one or two derived from calf spleen, Bos taurus, Escherichia coli, Salmonella typhimurium, Bacillus cereus, Bacillus clausii, Aeromonas hydrophila, Bovine Salmonella enterica, Bacteroides fragilis, Deinococcus radiodurans, Aeromonas hydrophila more than one purine nucleoside phosphorylase.
- the PNP enzyme comprises derived from calfspleen, Bos taurus (Bovine), Escherichia coli K12, Salmonella typhimurium (strain ATCC 700720), Bacillus cereus (strain ATCC 14579), Bacillus clausii (strain KSM-K16), Aeromonas hydrophila, One or more purine nucleoside phosphorylases from Bovine Salmonella enterica, Bacteroides fragilis (strain ATCC 25285), Deinococcus radiodurans (strain ATCC 13939), Aeromonas hydrophila.
- D-ribose-1-phosphate and nicotinamide ribose in the present invention requires the participation of PNP enzyme, wherein the process of generating D-ribose-1-phosphate is not limited to the source of the PNP enzyme, as long as it belongs to the PNP enzyme of EC 2.4.2.1,
- the process of generating nicotinamide ribose requires specific PNP to complete, and the PNP enzymes provided in the present invention can all complete a two-step reaction.
- the enzyme composition for preparing ⁇ -nicotinamide mononucleotide uses adenosine and nicotinamide as raw materials to synthesize NMN through PNP enzyme and NRK enzyme, and NMN can be obtained only by the participation of two enzymes.
- the raw material cost of glycosides and nicotinamide is low, and the by-product adenine can be recycled and reused, which greatly reduces the cost.
- only 1 molecule of ATP needs to be consumed to generate 1 molecule of NMN, which greatly reduces the cost.
- What is more different from the conventional enzyme catalysis method is that the final step of NRK enzyme-catalyzed synthesis of NMN is irreversible, which can greatly improve the substrate conversion rate and further reduce the production cost.
- the present invention also provides the application of the enzyme composition in preparing an enzyme preparation for synthesizing ⁇ -nicotinamide mononucleotide or in synthesizing ⁇ -nicotinamide mononucleotide.
- the present invention also provides a method for synthesizing ⁇ -nicotinamide mononucleotide by enzymatic catalysis, using adenosine, nicotinamide, ATP, Mg ion or Mn ion, and phosphate as raw materials.
- the ⁇ -nicotinamide mononucleotide and phosphate are synthesized under the enzymatic catalysis of the enzyme composition; the phosphate can be recycled to the next reaction process.
- the reaction can generally be carried out in a buffer, such as Tris or PBS buffer.
- PBS buffer can be used as a reaction medium or as a phosphate in the reaction to initiate the reaction.
- the magnesium ion or manganese ion as a coenzyme factor can be provided by one or more of magnesium chloride, magnesium sulfate, magnesium sulfite, magnesium nitrate, manganese chloride, manganese sulfate, and manganese nitrate.
- the enzyme composition participates in the enzyme catalyzed reaction in the form of a host cell expressing each enzyme, an enzyme solution of each enzyme, or an immobilized enzyme of each enzyme.
- the host cell for expressing each enzyme is Escherichia coli containing a vector for expressing each enzyme, and the vector may express one of the enzymes alone or co-express two enzymes. Its specific preparation process is as follows:
- Extract bacterial strain DNA take it as a template, amplify the DNA fragment of target PNP enzyme or NRK enzyme by PCR (can be optimized according to the codon preference of Escherichia coli), Table 1 of the present invention lists the PNP used in the specific embodiment The sequence of the enzyme or NRK enzyme.
- the amplified gene was digested correspondingly by the restriction enzyme site, and then connected to the vector plasmid with the same restriction enzyme digestion; the correct plasmid was verified by gene sequencing and then transferred into E.
- the carrier plasmid is a commercially available pET28a plasmid, and each enzyme amplification primer can be designed according to each enzyme coding sequence.
- the enzyme liquid of each enzyme is an enzyme liquid extracted from host cells expressing each enzyme; the method is to collect the crude protein-containing supernatant from the collected wet cells through high-pressure crushing and high-speed centrifugation
- the solution is the enzyme solution containing the enzyme, and the supernatant can also be further purified.
- the present invention also includes the purification step of ⁇ -nicotinamide mononucleotide:
- the pure product of ⁇ -nicotinamide mononucleotide is obtained after filtration, chromatographic separation, concentration, crystallization and drying.
- the conversion rate of the substrate (calculated by adenosine, the molar ratio of the product to adenosine) is 90-99%, and the purity of the obtained NMN finished product is over 99%.
- the present invention uses adenosine and nicotinamide as raw materials, generates D-ribose-1-phosphate and nicotinamide ribose intermediates under the enzymatic catalysis of the enzyme composition of PNP enzyme and NRK enzyme, and finally obtains NMN;
- the entire reaction system only needs the participation of two enzymes, and the by-product adenine can be recycled and reused.
- only one molecule of ATP needs to be consumed when generating one molecule of NMN, which greatly reduces the process cost.
- the final step of NRK enzyme-catalyzed synthesis of NMN is irreversible, which can greatly improve the substrate conversion rate and further reduce the production cost.
- the present invention discloses an enzyme composition for preparing beta-nicotinamide mononucleotide and its application. Those skilled in the art can learn from the content of this article and appropriately improve process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
- the enzyme composition of the present invention and its application have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations to the enzyme composition and its application without departing from the content, spirit and scope of the present invention. Implement and apply the technology of the present invention.
- the synthetic method and the schematic diagram of the reaction principle of the present invention are intended to clearly describe the core reaction route, and do not limit whether the entire reaction is carried out by a one-step method or a multi-step method.
- the enzymes used in the present invention can be artificially synthesized according to the sequence, or the expression genes of each enzyme can be carried by a plasmid vector according to the method provided by the present invention, and the expression can be induced by the host cell.
- the present invention utilizes LX-1000EP epoxy resin (Xi'an Lanxiao Company) for one-time mixing and immobilization.
- LX-1000EP epoxy resin Xi'an Lanxiao Company
- the present invention utilizes LX-1000EP epoxy resin (Xi'an Lanxiao Company) for one-time mixing and immobilization.
- PNP pure enzyme liquid 100ml Xi'an Lanxiao Company
- NRK pure enzyme liquid 10ml mixes, add 80 grams of LX-1000EP epoxy resin, filter out the resin after stirring at room temperature for 8 hours, Wash three times with 25mM pH 8.0 potassium phosphate buffer, suction filtration to obtain immobilized enzyme, determine the immobilized enzyme PNP enzyme activity is 800U/g, NRK enzyme activity is 80U/g.
- the consumption of each reaction material can be adjusted according to the actual situation, in order to maximize the efficiency, the present invention provides the following concentrations and consumption of each reaction material:
- the ratio of PNP enzyme to NRK enzyme is calculated by the enzyme activity, and the enzyme activity ratio of PNP:NRK is 1:(0.01-100), preferably 1:(0.1-10).
- the pH of the reaction solution is maintained between 6.5-9.5, preferably 7.0-8.5; the reaction temperature is between 25-50°C, preferably 35-45°C;
- PNP enzyme The protein sequence of PNP enzyme derived from Bos taurus is shown in attached table 1, and the corresponding DNA gene sequence was synthesized in vitro by Anhui General Organism after codon optimization. ), and ligated to the same digested pET28a plasmid (purchased from Addgene). The constructed plasmid was transferred into E.coli BL (DE3) strain (Shanghai Weidi Biology), and the correct colony was confirmed to be inoculated into the LB medium containing 100uM kanamycin.
- the composition of LB medium was: 1% Tryptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium hydrogen phosphate, 1% potassium dihydrogen phosphate, and 5% glycerol; when cells grow to mid-late logarithm, add 0.2 mM isopropyl- ⁇ -D - Thiogalactopyranoside (IPTG) induced protein expression at 30°C for 5 hours, and the wet cells were collected by centrifugation.
- IPTG isopropyl- ⁇ -D - Thiogalactopyranoside
- NRK enzyme extract the DNA of Haemophilus influenzae ATCC 51907 strain, use it as a template, and amplify the NadR gene fragment (genbank: NZ_CP009610.1) by PCR, (NadR sense primer: F: 5'-catatgcgagctaagtataacgcaaaat-3', NadR Antisense primer: R: 5'-ctcgagtcattgagatgtccctttttataggaaaggt-3', synthesized by Jinweizhi Company), the protein sequence corresponding to this gene fragment is shown in attached table 1.
- the amplified gene was digested with Nde I/Xho I purchased by NEB Company, and connected to the pET28a plasmid (purchased from Addgene) of the same restriction enzyme cut, and the constructed plasmid was transferred into E.coli BL21 ( DE3) strain (Shanghai Weidi Bio), confirm that the correct colonies are cultured in LB medium containing 100uM kanamycin, the LB medium is composed of: 1% tryptone, 0.5% yeast powder, 1% NaCl, 1% % potassium dihydrogen phosphate, 1% potassium dihydrogen phosphate, and 5% glycerol; when cells grow to mid-to-late logarithmic stage, add 0.2 mM isopropyl- ⁇ -D-thiogalactopyranoside (IPTG) at 30 Protein expression was induced at °C for 5 hours, and wet cells were collected by centrifugation.
- IPTG isopropyl- ⁇ -D-thiogalactopyranoside
- the enzymatic activity of the PNP enzyme of the present invention is defined as: the amount of enzyme required to convert 1 micromol of adenosine to D-ribose-1-phosphate in 1 minute is one activity unit (U).
- the enzymatic activity of the NRK enzyme of the present invention is defined as: the amount of enzyme required to convert 1 micromol of nicotinamide ribose into ⁇ -nicotinamide ribose mononucleotide in 1 minute is one activity unit (U).
- the enzyme activity of the above PNP crude enzyme solution was determined to be 75 U/ml, and the enzyme activity of NRK was 600 U/ml.
- reaction tank prepare 150mM adenosine, 150mM nicotinamide, 20mM MgCl 2 , 150mM ATP, 20mM pH8.0 PBS buffer, adjust the pH value to 6.5, the total volume of the reaction solution is 0.8L, add the above PNP crude enzyme solution to 32ml, Add 4 ml of the above NRK crude enzyme solution, and react at 37 °C for 8 h to maintain pH 8.0 to generate NMN 135 mM (45.1 g/L), and the substrate conversion rate is 90%.
- PNPase 1 source Escherichia coli (strain K12);
- PNP enzyme 2 source Bacillus clausii strain KSM-K16
- PNP/NRK enzyme ratio 100:1, 35°C, pH8.5, substrate concentration of adenosine is 135mM, nicotinamide substrate concentration is 135mM, ATP substrate concentration is 135mM, magnesium ion concentration is 200mM, Tris concentration is It is 20mM, and the substrate conversion rate is 92%;
- PNP enzyme 1 derived from Escherichia coli (strain K12) PNP enzyme protein sequence is shown in attached table 1, the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then genetic recombination is carried out according to the method of Example 1 Bacterial construction and cell culture, and the wet cells were collected by centrifugation.
- PNP enzyme 2 The PNP enzyme protein sequence derived from acillus clausii (strain KSM-K16) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then according to the method of Example 1 The recombinant bacteria were constructed and the bacteria were cultured, and the wet bacteria were collected by centrifugation.
- NRK enzyme protein sequence derived from Salmonella typhimurium (ATCC 700720) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then genetic recombination is carried out according to the method of Example 1. Bacterial construction and cell culture, and the wet cells were collected by centrifugation.
- PNP enzyme source Bacteroides fragilis (strain ATCC 25285);
- the ratio of PNP to NRK enzymes is (10:1);
- PNP/NRK enzyme ratio 10:1, 40°C, pH 7.0
- Substrate concentration of adenosine 50mM, nicotinamide substrate concentration 50mM, ATP substrate concentration 80mM, manganese ion concentration 5mM, phosphate The concentration is 200mM, and the substrate conversion rate is 91.1%;
- PNP enzyme The protein sequence of PNP enzyme derived from Bacteroides fragilis (strain ATCC 25285) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then genetic recombination is carried out according to the method of Example 1. Bacterial construction and cell culture, and the wet cells were collected by centrifugation.
- NRK enzyme The protein sequence of NRK enzyme derived from B.pseudomallei K96243 is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then the construction of recombinant bacteria is carried out according to the method of Example 1. And the bacteria were cultured, and the wet bacteria were collected by centrifugation.
- the supernatant liquid containing crude protein was collected by high pressure crushing and centrifugation respectively.
- the supernatant was purified by nickel ion chelating affinity column (Yisheng Bio) to obtain PNP pure enzyme liquid and NRK pure enzyme liquid, of which the activity of PNP pure enzyme liquid was 6800U/ml, and the activity of NRK pure enzyme liquid was 6400U/ml.
- the obtained pure enzyme solution was fixed once by using LX-1000EP epoxy resin (Xi'an Lanxiao Company), and the method was as follows: 100ml of the above-mentioned PNP pure enzyme solution was added to 1L potassium phosphate buffer solution (1M pH7.5), NRK Mix 10 ml of pure enzyme solution, add 80 grams of LX-1000EP epoxy resin, stir at room temperature for 8 hours, filter out the resin, wash three times with 25mM pH 8.0 potassium phosphate buffer, and obtain immobilized enzyme after suction filtration, and measure the immobilized enzyme The PNP enzyme activity was 800U/g, and the NRK enzyme activity was 80U/g.
- Source of PNP enzyme Salmonella typhimurium (ATCC 700720);
- PNP/NRK enzyme ratio 1:100, 35°C, pH 7.5, substrate concentration of 100mM adenosine, 100mM substrate concentration of nicotinamide, 100mM substrate concentration of ATP, 20mM concentration of magnesium ion, phosphate The concentration is 5mM, and the substrate conversion rate is 99%;
- PNP enzyme The protein sequence of PNP enzyme derived from Salmonella typhimurium (ATCC 700720) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then genetic recombination is carried out according to the method of Example 1 Bacterial construction and cell culture, and the wet cells were collected by centrifugation.
- the PNP pure enzyme solution was prepared according to the method of Example 3, and the activity of the PNP pure enzyme solution was 6200 U/ml;
- NRKPNP co-expression + whole cell catalytic form enzyme activity ratio 1:1
- the protein sequence of the PNP enzyme derived from Aeromonas hydrophila is shown in Table 1.
- the corresponding DNA gene sequence was codon-optimized and synthesized in vitro by Anhui General Organisms, and BamH I/Not I was used for the corresponding enzyme digestion (NEB company).
- the NRK enzyme protein sequence derived from Saccharomyces cerevisiae ATCC 204508 is shown in Table 1.
- the corresponding DNA gene sequence was codon-optimized and synthesized in vitro by Anhui General Organisms, and BamH I/Not I was used to carry out the corresponding enzyme digestion (NEB Company) .
- the above-mentioned two end genes of the digested gene were simultaneously ligated to the same digested pRSFDuet-1 plasmid (purchased from Biowind).
- the constructed plasmid was transferred into E.coli BL21(DE3) strain (Shanghai Weidi Biology), and confirmed that the correct colony was cultured in LB medium containing 100uM kanamycin.
- the LB medium was composed of: 1% pancreatic Peptone, 0.5% yeast powder, 1% NaCl, 1% dipotassium phosphate, 1% potassium dihydrogen phosphate, and 5% glycerol; when cells grow to mid-logarithmic stage, add 0.2mM isopropyl- ⁇ -D- Protein expression was induced by thiogalactopyranoside (IPTG) at 30°C for 5 hours, and the wet cells were collected by centrifugation.
- IPTG thiogalactopyranoside
- the wet cells after the above centrifugation were washed twice with 20mM pH7.0 PBS buffer and resuspended, and 0.15% TritonX-100 was added for low-speed stirring for 30min, and the wet cells were collected by centrifugation.
- the PNP enzyme activity of the permeabilized wet cells was determined to be 750 U/g, and the NRK enzyme activity was 750 U/g.
- Source of PNPase 1 Deinococcus radiodurans (strain ATCC 13939);
- Source of PNPase 2 Homo sapiens (Human);
- NRK enzyme source Ashbya gossypii (strain ATCC 10895);
- Substrate concentration of adenosine is 100mM
- substrate concentration of nicotinamide is 100mM
- substrate concentration of ATP is 100mM
- concentration of magnesium ion is 20 mM
- the phosphate concentration is 20 mM
- the substrate conversion rate is 90%;
- Substrate concentration of adenosine is 100mM
- substrate concentration of nicotinamide is 100mM
- substrate concentration of ATP is 100mM
- concentration of magnesium ion is 20 mM
- the phosphate concentration is 20 mM
- the substrate conversion rate is 95%;
- PNPase 1/NRK enzyme ratio 1:0.518, immobilized enzyme, 25°C, pH 9.5
- Substrate concentration of adenosine 1mM, nicotinamide substrate concentration 1mM, ATP substrate concentration 1mM, magnesium ion The concentration is 5mM, the phosphate concentration is 5mM, and the substrate conversion rate is 99%;
- PNPase 1/NRK enzyme ratio 1:0.518, immobilized enzyme, 45°C, pH 9.5
- Substrate concentration of adenosine 1mM, nicotinamide substrate concentration 1mM, ATP substrate concentration 1mM, magnesium ion The concentration is 5mM, the phosphate concentration is 5mM, and the substrate conversion rate is 99%;
- PNP enzyme 1 The protein sequence of PNP enzyme derived from Deinococcus radiodurans (strain ATCC 13939) is shown in attached table 1, and the corresponding DNA gene sequence was synthesized in vitro by Anhui General Organism after codon optimization, and then carried out according to the method of Example 1. Recombinant bacteria were constructed and cultured, and the wet bacteria were collected by centrifugation.
- PNP enzyme 2 The protein sequence of PNP enzyme derived from Homo sapiens (Human) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then genetic recombination is carried out according to the method of Example 1. Bacterial construction and cell culture, and the wet cells were collected by centrifugation.
- NRK enzyme protein sequence derived from Ashbya gossypii (strain ATCC 10895) is shown in attached table 1, and the corresponding DNA gene sequence is synthesized in vitro by Anhui General Organism after codon optimization, and then the gene is carried out according to the method of Example 1.
- the recombinant bacteria were constructed and cultured, and the wet bacteria were collected by centrifugation.
- the enzyme activity of the crude enzyme solution of the above PNP enzyme 1 was determined to be 450 U/ml, the enzyme activity of the crude enzyme solution of PNP enzyme 2 was 550 U/ml, and the enzyme activity of the NRK crude enzyme solution was 760 U/ml.
- the PNP enzyme 1 and NRK crude enzyme solutions prepared by the above method were centrifuged to collect the supernatant, and the supernatant was purified with a nickel ion chelating affinity column (Yisheng Bio) to obtain the PNP enzyme 1 pure enzyme solution and the NRK pure enzyme solution.
- the activity of PNP enzyme 1 pure enzyme solution is 4800U/ml
- the activity of NRK pure enzyme solution is 6700U/ml.
- the pure enzyme liquid obtained above was fixed once using LX-1000EP epoxy resin (Xi'an Lanxiao Company), and the method was as follows: in 2L potassium phosphate buffer solution (1M pH7.5), add the above-mentioned PNP enzyme 1 pure enzyme liquid 50ml and 40ml of NRK pure enzyme solution were mixed well, added 160g LX-1000EP epoxy resin, stirred at room temperature for 8 hours, filtered out the resin, washed three times with 25mM pH 8.0 potassium phosphate buffer, and obtained the immobilized enzyme after suction filtration.
- the enzymatic activity of immobilized enzyme PNPase 1 was 98.4 U/g, and the enzymatic activity of NRK was 190 U/g.
- PNP enzyme 1 prepare 100 mM adenosine, 100 mM nicotinamide, 20 mM MgCl 2 , 100 mM ATP, 20 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 8.0, and the total volume of the reaction solution is 0.8L .
- the reaction solution was filtered, separated by chromatography, concentrated, crystallized and dried to obtain 18.04 g of pure NMN, with a purity of 99.1% of the NMN product.
- PNP enzyme 2 prepare 100 mM adenosine, 100 mM nicotinamide, 20 mM MgCl 2 , 100 mM ATP, 20 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 8.0, and the total volume of the reaction solution is 0.8L .
- the result data shows that the reaction can only proceed to the first step and cannot continue, or the efficiency is extremely low, which also shows the process of generating nicotinamide ribose. Requires specific PNP enzymes to do so.
- PNP enzyme 1 prepare 100 mM adenosine, 100 mM nicotinamide, 20 mM MgCl 2 , 100 mM ATP, 20 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 8.0, and the total volume of the reaction solution is 0.8L . 3.28ml of PNP enzyme 1 pure enzyme solution and 4.53ml of NRK pure enzyme solution prepared above were added, and the reaction was stirred at 37°C for 5h to maintain pH 8.0 to generate NMN 95mM (31.73g/L) with a substrate conversion rate of 95%. The reaction solution was filtered, separated by chromatography, concentrated, crystallized and dried to obtain 20.05 g of pure NMN, the purity of NMN product was 99.4%.
- Immobilized enzyme form (PNPase 1): prepare 100 mM adenosine, 100 mM nicotinamide, 20 mM MgCl 2 , 100 mM ATP, 20 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 8.0, and the total volume of the reaction solution is 0.8 L. 160 g of the above-prepared PNP enzyme 1 and NRK immobilized enzyme were added, and the reaction was carried out at 37°C for 5.5 h to maintain pH 8.0 to generate NMN 94 mM (31.39 g/L), and the substrate conversion rate was 94%. The reaction solution was filtered, separated by chromatography, concentrated, crystallized and dried to obtain 19.8 g of pure NMN, with a purity of 99.4% of the NMN product.
- Immobilized enzyme form (PNP enzyme 1, 25°C): prepare 1 mM adenosine, 1 mM nicotinamide, 5 mM MgCl 2 , 1 mM ATP, 5 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 9.5, and the total reaction solution The volume is 0.8L. 160 g of the above-prepared PNP enzyme 1 and NRK immobilized enzyme were added, and the reaction was carried out at 25°C for 5.5 h with stirring to maintain pH 9.5 to generate NMN 0.99 mM (0.33 g/L) with a substrate conversion rate of 99%. The reaction solution was filtered, separated by chromatography, concentrated, crystallized and dried to obtain 0.13 g of pure NMN, the purity of NMN product was 99.4%.
- Immobilized enzyme form (PNP enzyme 1, 45°C): prepare 1 mM adenosine, 1 mM nicotinamide, 5 mM MgCl 2 , 1 mM ATP, 5 mM pH8.0 PBS buffer in a 1L reaction tank, adjust the pH to 9.5, and the total reaction solution The volume is 0.8L. 160 g of the above-prepared PNP enzyme 1 and NRK immobilized enzyme were added, and the reaction was stirred at 45°C for 0.5 h to maintain pH 9.5 to generate NMN 0.99 mM (0.33 g/L) with a substrate conversion rate of 97%. The reaction solution was filtered, separated by chromatography, concentrated, crystallized and dried to obtain 0.13 g of pure NMN, the purity of NMN product was 99.4%.
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Abstract
Description
Claims (8)
- 一种用于制备β-烟酰胺单核苷酸的酶组合物,其特征在于,由EC编号为EC 2.4.2.1的嘌呤核苷磷酸化酶和EC编号为EC 2.7.1.22烟酰胺核糖激酶组成;其中,所述嘌呤核苷磷酸化酶包含来源于calf spleen,Bos taurus,Escherichia coli,Salmonella typhimurium,Bacillus cereus,Bacillus clausii,Aeromonas hydrophila,Bovine Salmonella enterica,Bacteroides fragilis,Deinococcus radiodurans,Aeromonas hydrophila中的一种或两种以上嘌呤核苷磷酸化酶。
- 根据权利要求1所述酶组合物,其特征在于,所述烟酰胺核糖激酶来源于B.pseudomallei,Ashbya gossypii,Haemophilus influenzae,Saccharomyces cerevisiae,Schizosaccharomyces pombe,Salmonella typhimurium,Cupriavidus metallidurans,Xanthomonas campestris pv.Campestris,Agrobacterium vitis,Pseudarthrobacter chlorophenolicus,Actinobacillus succinogenes,Homo sapiens,Saccharomyces cerevisiae,Ashbya gossypii中的一种或两种以上。
- 权利要求1-2任意一项所述酶组合物在制备合成β-烟酰胺单核苷酸的酶制剂中的应用或在合成β-烟酰胺单核苷酸中的应用。
- 一种酶催化法合成β-烟酰胺单核苷酸的方法,其特征在于,以腺苷、烟酰胺、ATP、选自Mg离子和Mn离子之一的离子,以及磷酸盐为原料,在权利要求1-2任意一项所述酶组合物的酶催化作用下合成β-烟酰胺单核苷酸。
- 根据权利要求4所述方法,其特征在于,所述酶组合物以表达各酶的宿主细胞、各酶的酶液或各酶的固定化酶形式参与酶催化反应。
- 根据权利要求5所述方法,其特征在于,所述表达各酶的宿主细胞为含有表达各酶的载体或共表达两种酶的载体的大肠杆菌。
- 根据权利要求5所述方法,其特征在于,所述各酶的酶液为从表达各酶的宿主细胞中提取的酶液。
- 根据权利要求4所述方法,其特征在于,还包括β-烟酰胺单核苷 酸的纯化步骤:经过滤、层析分离,浓缩结晶干燥后获得β-烟酰胺单核苷酸的纯品。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1111061A (zh) * | 1993-12-14 | 1995-11-01 | 奥佩西克·戈德纳 | 嗜热细菌用于制造三唑核苷类的用途 |
US20170121746A1 (en) * | 2015-10-28 | 2017-05-04 | The Procter & Gamble Company | Method For Preparing Nicotinamide Riboside |
CN111748537A (zh) * | 2020-08-04 | 2020-10-09 | 浙江华睿生物技术有限公司 | 一种尿苷磷酸酶突变体及其应用 |
CN112601543A (zh) * | 2018-07-09 | 2021-04-02 | 科德克希思公司 | 工程化嘌呤核苷磷酸化酶变体酶 |
CN112980906A (zh) * | 2021-04-14 | 2021-06-18 | 深圳瑞德林生物技术有限公司 | 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1111061A (zh) * | 1993-12-14 | 1995-11-01 | 奥佩西克·戈德纳 | 嗜热细菌用于制造三唑核苷类的用途 |
US20170121746A1 (en) * | 2015-10-28 | 2017-05-04 | The Procter & Gamble Company | Method For Preparing Nicotinamide Riboside |
CN112601543A (zh) * | 2018-07-09 | 2021-04-02 | 科德克希思公司 | 工程化嘌呤核苷磷酸化酶变体酶 |
CN111748537A (zh) * | 2020-08-04 | 2020-10-09 | 浙江华睿生物技术有限公司 | 一种尿苷磷酸酶突变体及其应用 |
CN112980906A (zh) * | 2021-04-14 | 2021-06-18 | 深圳瑞德林生物技术有限公司 | 一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用 |
Non-Patent Citations (2)
Title |
---|
LIAO YI-BO, WU MIN-HUI, LING SHU-LI, YING LIN: "Expression of Nicotinamide Phosphoribosyltransferase in Escherichia coli and Catalytic Synthesis of Nicotinamide Mononucleotide", MODERN FOOD SCIENCE AND TECHNOLOGY, HUANAN LIGONG DAXUE,SOUTH CHINA UNVERSITY OF TECHNOLOGY, CN, vol. 37, no. 2, 20 February 2021 (2021-02-20), CN , XP055977588, ISSN: 1673-9078, DOI: 10.13982/j.mfst.1673-9078.2021.2.0239 * |
WIELGUS-KUTROWSKA BEATA, E KULIKOWSKA, J WIERZCHOWSKI, A BZOWSKA, D SHUGAR: "Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases", EUROPEAN JOURNAL OF BIOCHEMISTRY, vol. 243, 15 January 1997 (1997-01-15), pages 408 - 414, XP055977591, DOI: 10.1111/j.1432-1033.1997.0408a.x * |
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