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 PDF

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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
pnp
nrk
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于铁妹
何秀秀
秦国富
凌瑞枚
林立峰
谭文静
潘俊锋
刘建
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Shenzhen Readline Biotech Co Ltd
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Shenzhen Readline Biotech Co Ltd
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Priority to AU2021440288A priority patent/AU2021440288B2/en
Priority to US18/555,029 priority patent/US20240279699A1/en
Priority to EP21936697.8A priority patent/EP4324927A4/en
Publication of WO2022217827A1 publication Critical patent/WO2022217827A1/zh
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    • 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

本发明涉及生物技术领域，公开了一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用。本发明以腺苷、烟酰胺为原料，在PNP酶和NRK酶的酶组合物的酶催化下生成D-核糖-1-磷酸和烟酰胺核糖中间体，最后获得NMN；整个反应体系仅需要2种酶的参与，副产物腺嘌呤可回收再利用。同时，生成1分子的NMN的同时只需要消耗1分子的ATP，大幅降低了工艺成本。最后一步NRK酶催化合成NMN反应不可逆，可大幅提高底物转化率，进一步降低生产成本。

Description

一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用
本申请要求于2021年04月14日提交中国专利局、申请号为202110397619.0、发明名称为“一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及生物技术领域，具体涉及一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用。
背景技术
β-烟酰胺单核苷酸(Nicotinamide mononucleotide,缩写成NMN)是生物体内存在的一种物质，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)。2017年3月David Scinclair研究团队发表在《science》上的一项研究表明，NAD+在小鼠体内的增加，使得大龄小鼠的组织和肌肉衰老迹象被逆转，这表明人类返老还童不再是梦想。由于NAD+分子量过大，无法通过口服摄取至细胞内，其体内主要依赖于细胞的合成，而且合成量很低。但随着对NAD+前体小分子物质NMN的研究发现，食用β-NMN可以有效提升体内NAD+的含量升高，并显著抑制衰老引起的新陈代谢，使得β-NMN成为了“不老神药”。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。
目前合成NMN的主要方法包括：化学合成、生物催化法。其中化学法成本高，同时造成严重的环境污染，已逐渐被生物催化法取代。相比较而言，生物酶法催化生产NMN更加高效、成本更低、节能环保。目前，有三种生物催化方法生产NMN，第一种是以烟酰胺核糖为原料，通过烟酰胺核糖激酶(Ribosylnicotinamide kinase，EC 2.7.1.22)在ATP供应下生成NMN。第二种是以烟酰胺、核糖和ATP为底物，经D-核糖激酶 (Ribokinase，EC2.7.1.15)、核酸磷酸焦磷酸激酶(ribose phosphate pyrophosphokinase，EC2.7.6.1)、和烟酰胺核糖磷酸转移酶(Nicotinamide phosphoribosyltransferase，EC.2.4.2.12)催化反应生成NMN。第三种是以腺苷或AMP、ATP、烟酰胺为原料，经腺苷激酶(EC 2.7.1.20)(以AMP为原料时不需要此酶)、腺嘌呤磷酸核糖转移酶(EC 2.4.2.7)、烟酰胺磷酸核糖转移酶(Nicotinamide phosphoribosyltransferase，EC.2.4.2.12)催化生成NMN。
上述第一种方法直接以烟酰胺核糖为原料，底物转化率高，但烟酰胺核糖价格昂贵，成本不具有优势。第二种、第三种方法最后均以PRPP和烟酰胺通过烟酰胺磷酸核糖转移酶(Nicotinamide phosphoribosyltransferase，EC.2.4.2.12)制备NMN。因烟酰胺磷酸核糖转移酶催化可逆反应，合成NMN的同时也能水解NMN，反应转化率较低。同时，中间体PRPP化合物不稳定，不利于反应进行。再次，上述两种方法多步反应均需要ATP参与反应，整条工艺路线需要消耗大量的ATP，导致该生物催化法的生产成本仍较高。有必要开发一种更为高效、低成本的生物催化制备NMN的新方法。
发明内容
有鉴于此，本发明的目的在于提供一种用于制备β-烟酰胺单核苷酸(NMN)的酶组合物，使其以腺苷、烟酰胺为原料合成β-烟酰胺单核苷酸时，能够在生成1分子的NMN的同时只需要消耗1分子的ATP，并且反应中最后一步合成NMN的反应不可逆，可大幅提高底物转化率，降低生产成本；
本发明的另外一个目的在于提供利用上述酶组合物合成NMN的方法以及提供上述酶组合物在合成NMN方面的相关应用。
为实现上述目的，本发明提供如下技术方案：
一种用于制备β-烟酰胺单核苷酸的酶组合物，包括EC编号为EC 2.4.2.1的嘌呤核苷磷酸化酶(purine-nucleoside phosphorylase，简写PNP酶)和EC编号为EC 2.7.1.22烟酰胺核糖激酶(ribosylnicotinamide kinase， 简写NRK酶)。
本发明针对目前的酶催化法的诸多缺陷，利用酶促反应技术提供了另外一种以腺苷、烟酰胺合成β-烟酰胺单核苷酸的工艺，反应过程如下式：
Figure PCTCN2021117960-appb-000001
腺苷和磷酸盐在PNP酶的作用下先合成D-核糖-1-磷酸，然后继续在该酶催化下和烟酰胺合成烟酰胺核糖，然后消耗一分子ATP在NRK酶的催化下不可逆反应生成NMN。
在上述反应过程中，PNP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的嘌呤核苷磷酸化酶。更优选地，所述PNP酶包含来源于calfspleen，Bos taurus(Bovine)，Escherichia coli K12,Salmonella typhimurium(strainATCC 700720)，Bacillus cereus(strain ATCC 14579)，Bacillus clausii(strain KSM-K16)，Aeromonas hydrophila，Bovine Salmonella enterica，Bacteroides fragilis(strain ATCC 25285)，Deinococcus radiodurans(strain ATCC 13939)，Aeromonas hydrophila一种或一种以上的嘌呤核苷磷酸化酶。
本发明生成D-核糖-1-磷酸以及烟酰胺核糖都需要PNP酶参与，其中生成D-核糖-1-磷酸的过程不对PNP酶的来源限定，只要属于EC 2.4.2.1的PNP酶即可，而生成烟酰胺核糖的过程需要特定PNP才能够完成，在本发明中提供的PNP酶均可以完成两步反应。
NRK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。更优选地，所述NRK酶来源于B.pseudomallei K96243，Ashbya gossypiiATCC 10895，Haemophilus influenzae ATCC 51907，Saccharomyces cerevisiae ATCC 204508，Schizosaccharomyces pombe ATCC 24843，Salmonella typhimurium(strain ATCC 700720)，Cupriavidus metallidurans(strain ATCC 43123，Deinococcus radiodurans(strain ATCC 13939)，Xanthomonas campestris pv.campestris(strain ATCC 33913)，Agrobacterium vitis(strain ATCC BAA-846)(Rhizobium vitis(strain S4))，Pseudarthrobacter chlorophenolicus，Actinobacillus succinogenes(strain ATCC 55618)，Homo sapiens(Human)，Saccharomyces cerevisiae(strain ATCC 204508)(Baker's yeast)，Ashbya gossypii(strain ATCC 10895)中的一种或两种以上。
本发明提供的用于制备β-烟酰胺单核苷酸的酶组合物，以腺苷、烟酰胺为原料经过PNP酶、NRK酶合成NMN，仅需要2种酶的参与即可获得NMN，腺苷、烟酰胺原料成本低廉，副产物腺嘌呤可回收再利用，大幅降低成本。同时，生成1分子的NMN的同时只需要消耗1分子的ATP，大幅降低了成本。更为与常规酶催化法不同的是，最后一步NRK酶催化合成NMN反应不可逆，可大幅提高底物转化率，进一步降低生产成本。
因此，本发明还提供了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。
依据应用，本发明还提供了一种酶催化法合成β-烟酰胺单核苷酸的方法，以腺苷、烟酰胺、ATP、Mg离子或Mn离子、磷酸盐为原料，在本发明所述酶组合物的酶催化作用下合成β-烟酰胺单核苷酸和磷酸盐；磷酸盐可循环利用到下一反应过程中。反应一般可在缓冲液中进行，如Tris或PBS缓冲液，为了方便高效的进行反应，采用PBS缓冲液既可以 充当反应介质，也可以作为反应中的磷酸盐启动反应。
作为优选，作为辅酶因子的镁离子或锰离子可由氯化镁、硫酸镁、亚硫酸镁、硝酸镁、氯化锰、硫酸锰、硝酸锰中的一种或多种提供。
作为优选，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。在本发明具体实施方式中，所述表达各酶的宿主细胞为含有表达各酶的载体的大肠杆菌，所述载体可以是单独表达其中一种酶，也可以是共表达两种酶。其具体的制备过程如下：
提取菌株DNA，以其为模板，通过PCR扩增出目标PNP酶或NRK酶的DNA片段(可按照大肠杆菌密码子偏好性进行优化)，本发明表1列举了具体实施例中所使用的PNP酶或NRK酶的序列。利用酶切位点对扩增后的基因进行相应的酶切，并连接到相同酶切的载体质粒上；通过基因测序验证正确的质粒进而转入E.coli BL21(DE3)菌株中,于LB培养液中进行培养，然后IPTG条件下诱导表达,收集各酶的湿细胞；所述载体质粒为市售的pET28a质粒，各酶扩增引物可根据各酶编码序列进行设计。
在本发明具体实施方式中，所述各酶的酶液为从表达各酶的宿主细胞中提取的酶液；方法为将所收集的湿细胞经过高压破碎、高速离心收集含粗蛋白的上清液，即为含酶的酶液，也可以对上清液进一步纯化。
作为优选，本发明还包括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。
按照本发明所述方法生成NMN，底物转化率(以腺苷计算，产物和腺苷的摩尔比)为90-99％，获得的NMN成品纯度达99％以上。
由以上技术方案可知，本发明以腺苷、烟酰胺为原料，在PNP酶和NRK酶的酶组合物的酶催化下生成D-核糖-1-磷酸和烟酰胺核糖中间体，最后获得NMN；整个反应体系仅需要2种酶的参与，副产物腺嘌呤可回收再利用。同时，生成1分子的NMN的同时只需要消耗1分子的ATP，大幅降低了工艺成本。最后一步NRK酶催化合成NMN反应不可逆，可大幅提高底物转化率，进一步降低生产成本。
具体实施方式
本发明公开了一种用于制备β-烟酰胺单核苷酸的酶组合物及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明酶组合物及其应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文酶组合物及其应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明所述合成方法及其反应原理示意图旨在清楚的描述核心的反应路线，并不限制整个反应采用一步法还是多步法进行。
本发明所采用的各酶可以根据序列进行人工合成，也可按照本发明提供的方法通过质粒载体搭载各酶表达基因，借助宿主细胞诱导表达。
在进行固定化时，可参照本领域常规的固定化酶制备方式，在本发明具体实施方式中，本发明利用LX-1000EP环氧树脂(西安蓝晓公司)进行一次性混合固定，其固定方法如下:在1L磷酸钾缓冲液(1M pH7.5)中加入上述PNP纯酶液100ml，NRK纯酶液10ml混均，加入80克LX-1000EP环氧树脂，室温搅拌8小时后过滤出树脂,用25mM pH 8.0磷酸钾缓冲液洗涤三次，抽滤后得固定化酶，测定该固定化酶PNP酶活力为800U/g，NRK酶活力为80U/g。
依照本发明工艺的反应路线，各反应物质的用量可以根据实际情况调整，为了最大效率化，本发明提供了如下各反应物质的浓度和用量：
PNP酶与NRK酶的比例以酶活计，PNP：NRK的酶活比例为1：(0.01～100)，优选的为1：(0.1～10)。腺苷的底物浓度为1-150mM，烟酰胺的底物浓度为1-150mM，ATP的底物浓度为1mM-150mM，镁离子或锰离子的浓度为5mM-200mM，磷酸盐或Tris的浓度为5-200mM。
反应过程维持反应液的pH在6.5-9.5之间，优选7.0-8.5；反应温度在25-50℃之间，优选35-45℃；
本发明具体实施例中所使用的酶的相关信息参见表1和表2：
表1
Figure PCTCN2021117960-appb-000002
Figure PCTCN2021117960-appb-000003
Figure PCTCN2021117960-appb-000004
表2
Figure PCTCN2021117960-appb-000005
Figure PCTCN2021117960-appb-000006
下面结合实施例，进一步阐述本发明。
实施例1：
PNP酶来源Bos taurus(Bovine)；
NRK酶的制备来源Haemophilus influenzae(ATCC 51907)；
PNP/NRK酶比例1:1，37℃，pH6.5腺苷的底物浓度为150mM，烟酰胺的底物浓度为150mM，ATP的底物浓度为150mM，镁离子的浓度为20mM，磷酸盐浓度为20mM、底物转化率90％；
PNP酶的制备：来源于Bos taurus的PNP酶蛋白质序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，利用Nde I/Xho I进行相应的酶切(NEB公司)，并连接到相同酶切的pET28a质粒上(购于Addgene)。将构建好的质粒转入E.coli BL(DE3)菌株中(上海唯地生物)，确认正确的菌落接种至到含100uM卡那霉素的LB培养液中，LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸二氢钾以及5％的甘油；当细胞增长至对数中后期，加入0.2mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在30℃诱导蛋白表达5小时，离心收集湿菌体。
NRK酶的制备：提取Haemophilus influenzae ATCC 51907菌株DNA，以其为模板，通过PCR扩增出NadR基因片段(genbank:NZ_CP009610.1)，(NadR正义引物：F:5’-catatgcgagctaagtataacgcaaaat-3’，NadR反义引物：R:5’-ctcgagtcattgagatgtcccttttataggaaaggt-3’，由金唯智公司合成)，该基因片段对应的蛋白质序列如附表1所示。利用NEB公司购买的Nde I/Xho I对扩增后的基因进行相应的酶切，并连接到相同酶切的pET28a质粒上(购于Addgene),将构建好的质粒转入E.coli BL21(DE3)菌株中(上海唯地生物)，确认正确的菌落培养到含100uM卡那霉素的LB培养液中，LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸二氢钾以及5％的甘油；当细胞增长至对数中后期，加入0.2mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在30℃诱导蛋白表达5小时，离心收集湿细胞。
分别称取上述PNP酶、NRK酶各100g湿细胞，分别用20mM Tris pH  7.5 1000ml水溶液重悬后经高压破碎得PNP酶及NRK酶的粗酶液。使用现有技术记载的公知的测定PNP酶及NRK酶活性的方法，以在特定条件下，1分钟内转化1微摩尔底物所需的酶量为一个活力单位(U)。本发明PNP酶的酶活定义为：1分钟转化1微摩尔腺苷生成D-核糖-1-磷酸所需的酶量为一个活力单位(U)。本发明NRK酶的酶活定义为：1分钟转化1微摩尔烟酰胺核糖生成β-烟酰胺核糖单核苷酸所需的酶量为一个活力单位(U)。测定上述PNP粗酶液酶活力为75U/ml，NRK酶活力为600U/ml。
在1L反应罐中配制150mM腺苷，150mM烟酰胺，20mM MgCl 2，150mM ATP，20mM pH8.0PBS缓冲液，调节pH值为6.5反应液总体积为0.8L，加入上述PNP粗酶液为32ml，加入上述NRK粗酶液为4ml，37℃搅拌下反应8h维持pH8.0，生成NMN 135mM(45.1g/L)，底物转化率为90％，反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品27.1g，NMN产品纯度99.2％，NMN产品核磁结果为： 1H NMR(400MHz,D 2O)δ9.46(s,1H),9.29(d,J＝5.9Hz,1H),8.99(d,J＝7.9Hz,1H),8.31(t,J＝7.1Hz,1H),6.22(d,J＝5.1Hz,1H),4.64(s,1H),4.58-4.55(m,1H),4.46-4.43(m,1H),4.32-4.29(m,1H),4.17-4.13(m,1H)。
实施例2：
PNP酶1来源Escherichia coli(strain K12)；
PNP酶2来源Bacillus clausii(strain KSM-K16)；
NRK酶来源Salmonella typhimurium(ATCC 700720)；
PNP/NRK酶比例100：1，35℃，pH8.5腺苷的底物浓度为135mM，烟酰胺的底物浓度为135mM，ATP的底物浓度为135mM，镁离子的浓度为200mM，Tris浓度为20mM、底物转化率92％；
PNP酶1的制备：来源于Escherichia coli(strain K12)PNP酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
PNP酶2的制备：来源于acillus clausii(strain KSM-K16)的PNP酶蛋 白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
NRK酶的制备：来源于Salmonella typhimurium(ATCC 700720)的NRK酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
分别称取上述PNP酶1、PNP酶2、NRK酶各100g湿细胞，分别用20mM Tris pH 7.5 1000ml水溶液重悬后经高压破碎得PNP酶1、PNP酶2及NRK酶的粗酶液。测定上述PNP酶1粗酶液酶活力为200U/ml、PNP酶2粗酶液酶活力为500U/ml，NRK酶活力为50U/ml。
在1L反应罐中配制135mM腺苷，135mM烟酰胺，200mM MgCl 2，135mM ATP，20mM Tris，调节pH值为8.5，反应液总体积为0.8L，加入上述PNP酶1粗酶液为50ml，PNP酶2粗酶液为30ml，加入上述NRK粗酶液为5ml，35℃搅拌下反应24h维持pH8.5，生成NMN 124.2mM(41.5g/L),底物转化率92％，反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品28.2g，NMN产品纯度99.3％。
实施例3：
PNP酶来源Bacteroides fragilis(strain ATCC 25285)；
NRK酶来源B.pseudomallei K96243；
固定化酶形式；
PNP与NRK酶的比例为(10：1)；
PNP/NRK酶比例10:1，40℃，pH7.0腺苷的底物浓度为50mM，烟酰胺的底物浓度为50mM，ATP的底物浓度为80mM，锰离子的浓度为5mM，磷酸盐浓度为200mM、底物转化率91.1％；
PNP酶的制备：来源于Bacteroides fragilis(strain ATCC 25285)PNP酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养， 离心收集湿菌体。
NRK酶的制备：来源于B.pseudomallei K96243的NRK酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
上述收集的含PNP酶，NRK酶菌体细胞混合后，分别经过高压破碎、离心收集含粗蛋白的上清液。上清液分别用镍离子螯合亲和柱(翊圣生物)纯化获得PNP纯酶液及NRK纯酶液，其中PNP纯酶液活力为6800U/ml，NRK纯酶液活力6400U/ml。
将获得的纯酶液，利用LX-1000EP环氧树脂(西安蓝晓公司)进行一次固定，其方法如下：在1L磷酸钾缓冲液(1M pH7.5)中加入上述PNP纯酶液100ml，NRK纯酶液10ml混均，加入80克LX-1000EP环氧树脂，室温搅拌8小时后过滤出树脂,用25mM pH 8.0磷酸钾缓冲液洗涤三次，抽滤后得固定化酶，测定该固定化酶PNP酶活力为800U/g，NRK酶活力为80U/g。
在1L反应罐中配制50mM腺苷，50mM烟酰胺，5mM MnCl 2，80mM ATP，200mM pH7.0PBS缓冲液，调节pH值为7.0，反应液总体积为0.8L，加入上述固定化酶20g,40℃搅拌下反应8h维持pH7.0，生成NMN 45.5mM(15.2g/L)，底物转化率为91.1％，反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品9.12g，NMN产品纯度99.2％。
实施例4：
PNP酶来源：Salmonella typhimurium(ATCC 700720)；
NRK酶来源B.pseudomallei K96243纯化酶；
PNP/NRK酶比例1：100，35℃，pH7.5腺苷的底物浓度为100mM，烟酰胺的底物浓度为100mM，ATP的底物浓度为100mM，镁离子的浓度为20mM，磷酸盐浓度为5mM、底物转化率99％；
PNP酶的制备：来源于Salmonella typhimurium(ATCC 700720)的PNP酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生 物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。按实施例3的方法制备获得PNP纯酶液，PNP纯酶液活力为6200U/ml；
在1000ml三角瓶中配制100mM腺苷，100mM烟酰胺，20mM MgCl 2，100mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为500ml，加入上述的PNP纯酶液4.8ml，加入来源B.pseudomallei K96243的NRK纯酶液46ml，35℃搅拌下反应24h维持pH8.0，生成NMN 99mM(30.07g/L)，底物转化率99％，反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品12.7g，NMN产品纯度99.4％。
实施例5：
PNP酶来源Aeromonas hydrophila；
NRK酶来源Saccharomyces cerevisiae ATCC 204508；
NRKPNP共表达+全细胞催化形式(酶活比例1：1)；
PNP/NRK酶比例1：1，37℃，pH8.0腺苷的底物浓度为135mM，烟酰胺的底物浓度为135mM，ATP的底物浓度为135mM，镁离子的浓度为20mM，磷酸盐浓度为20mM、底物转化率92％；
全细胞制备：来源于Aeromonas hydrophila的PNP酶蛋白序列如表1所示，将对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，利用BamH I/Not I进行相应的酶切(NEB公司)。来源于Saccharomyces cerevisiae ATCC 204508的NRK酶蛋白序列如表1所示，将对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，利用BamH I/Not I进行相应的酶切(NEB公司)。将酶切后的基因上述两端基因同时连接在相同酶切的pRSFDuet-1质粒上(购于生物风)。将构建好的质粒转入E.coli BL21(DE3)菌株中(上海唯地生物)，确认正确的菌落培养到含100uM卡那霉素的LB培养液中，LB培养基构成为：1％胰蛋白胨、0.5％酵母粉，1％NaCl，1％磷酸氢二钾、1％磷酸二氢钾以及5％的甘油；当细胞增长至对数中后期，加入0.2mM异丙基-β-D-硫代吡喃半乳糖苷(IPTG)在30℃诱导蛋白表达5小时，离心收集湿细胞。
取上述离心后的湿细胞用20mM pH7.0PBS缓冲液洗涤二遍后重悬，加入0.15％TritonX-100低速搅拌处理30min，离心收集湿细胞。测定通透后的湿细胞PNP酶活力为750U/g，NRK酶活力为750U/g。
在1L反应罐中配制135mM腺苷，135mM烟酰胺，20mM MgCl 2，135mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为0.8L，加入上述湿细胞32g，37℃搅拌下反应12h维持pH8.0，生成NMN124.2mM(41.48g/L)，底物转化率为92％，反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品24.22g，NMN产品纯度99.1％。
实施例6：
PNP酶1来源：Deinococcus radiodurans(strain ATCC 13939)；
PNP酶2来源：Homo sapiens(Human)；
NRK酶来源：Ashbya gossypii(strain ATCC 10895)；
该实施例比较了相同酶活下(1：0.518)，不同形式粗酶、纯酶、固定化酶的效果：
PNP酶1/NRK酶比例1：0.518，粗酶，37℃，pH8.0腺苷的底物浓度为100mM，烟酰胺的底物浓度为100mM，ATP的底物浓度为100mM，镁离子的浓度为20mM，磷酸盐浓度为20mM、底物转化率90％；
PNP酶1/NRK酶比例1：0.518，纯酶，37℃，pH8.0腺苷的底物浓度为100mM，烟酰胺的底物浓度为100mM，ATP的底物浓度为100mM，镁离子的浓度为20mM，磷酸盐浓度为20mM、底物转化率95％；
PNP酶1/NRK酶比例1：0.518，固定化酶，37℃，pH8.0腺苷的底物浓度为100mM，烟酰胺的底物浓度为100mM，ATP的底物浓度为100mM，镁离子的浓度为20mM，磷酸盐浓度为20mM、底物转化率94％；
PNP酶1/NRK酶比例1：0.518，固定化酶，25℃，pH9.5腺苷的底物浓度为1mM，烟酰胺的底物浓度为1mM，ATP的底物浓度为1mM，镁离子的浓度为5mM，磷酸盐浓度为5mM、底物转化率99％；
PNP酶1/NRK酶比例1：0.518，固定化酶，45℃，pH9.5腺苷的底物浓度为1mM，烟酰胺的底物浓度为1mM，ATP的底物浓度为1mM，镁离子的浓度为5mM，磷酸盐浓度为5mM、底物转化率99％；
PNP酶1的制备：来源于Deinococcus radiodurans(strain ATCC 13939)的PNP酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
PNP酶2的制备：来源于Homo sapiens(Human)的PNP酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
NRK酶的制备：来源于Ashbya gossypii(strain ATCC 10895)的NRK酶蛋白序列见附表1，对应的DNA基因序列经密码子优化后由安徽通用生物体外合成，然后按实施例1的方法进行基因重组菌构建及菌体培养，离心收集湿菌体。
分别称取上述PNP酶1湿细胞、PNP酶2湿细胞、NRK酶湿细胞100g，分别用20mM Tris pH 7.5 1000ml水溶液重悬后经高压破碎获得PNP酶的粗酶液及NRK酶的粗酶液。测定上述PNP酶1的粗酶液酶活力为450U/ml，PNP酶2粗酶液酶活力为550U/ml，NRK粗酶液酶活力为760U/ml。
按上述方法制备的PNP酶1、NRK粗酶液经离心收集上清液，上清液分别用镍离子螯合亲和柱(翊圣生物)纯化获得PNP酶1纯酶液及NRK纯酶液，其中PNP酶1纯酶液活力为4800U/ml，NRK纯酶液活力6700U/ml。
将上述获得的纯酶液，利用LX-1000EP环氧树脂(西安蓝晓公司)进行一次固定，其方法如下：在2L磷酸钾缓冲液(1M pH7.5)中加入上述PNP酶1纯酶液50ml，NRK纯酶液40ml混均，加入160克LX-1000EP环氧树脂，室温搅拌8小时后过滤出树脂,用25mM pH 8.0磷酸钾缓冲液洗涤三次，抽滤后得固定化酶，测定该固定化酶PNP酶1酶活力为 98.4U/g，NRK酶活力为190U/g。
粗酶形式(PNP酶1)：在1L反应罐中配制100mM腺苷，100mM烟酰胺，20mM MgCl 2，100mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为0.8L。加入上述制备的PNP酶1粗酶液35ml、NRK粗酶液40ml，在37℃搅拌下反应6h维持pH8.0，生成NMN 90mM(30.06g/L)，底物转化率为90％。反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品18.04g，NMN产品纯度99.1％。
粗酶形式(PNP酶2)：在1L反应罐中配制100mM腺苷，100mM烟酰胺，20mM MgCl 2，100mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为0.8L。加入上述制备的PNP酶2粗酶液28.8ml、NRK粗酶液40ml，在37℃搅拌下反应6h维持pH8.0，生成NMN 1mM(0.33g/L)，底物转化率为1％，底物转化率过低，无法纯化获得纯品；
采用只能完成第一步反应无法完成第二步反应的PNP酶2，通过结果数据说明确实反应只能进行到第一步无法继续，或者效率极其低下，这也说明了生成烟酰胺核糖的过程需要特定PNP酶才能够完成。
纯酶形式(PNP酶1)：在1L反应罐中配制100mM腺苷，100mM烟酰胺，20mM MgCl 2，100mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为0.8L。加入上述制备的PNP酶1纯酶液3.28ml、NRK纯酶液4.53ml，在37℃搅拌下反应5h维持pH8.0，生成NMN 95mM(31.73g/L)，底物转化率为95％。反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品20.05g，NMN产品纯度99.4％。
固定化酶形式(PNP酶1)：在1L反应罐中配制100mM腺苷，100mM烟酰胺，20mM MgCl 2，100mM ATP，20mM pH8.0PBS缓冲液，调节pH值为8.0，反应液总体积为0.8L。加入上述制备PNP酶1、NRK固定化酶160g，在37℃搅拌下反应5.5h维持pH8.0，生成NMN 94mM(31.39g/L)，底物转化率为94％。反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品19.8g，NMN产品纯度99.4％。
固定化酶形式(PNP酶1，25℃)：在1L反应罐中配制1mM腺苷，1mM烟酰胺，5mM MgCl 2，1mM ATP，5mM pH8.0PBS缓冲液，调节 pH值为9.5，反应液总体积为0.8L。加入上述制备PNP酶1、NRK固定化酶160g，在25℃搅拌下反应5.5h维持pH9.5，生成NMN 0.99mM(0.33g/L)，底物转化率为99％。反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品0.13g，NMN产品纯度99.4％。
固定化酶形式(PNP酶1，45℃)：在1L反应罐中配制1mM腺苷，1mM烟酰胺，5mM MgCl 2，1mM ATP，5mM pH8.0PBS缓冲液，调节pH值为9.5，反应液总体积为0.8L。加入上述制备PNP酶1、NRK固定化酶160g，在45℃搅拌下反应0.5h维持pH9.5，生成NMN 0.99mM(0.33g/L)，底物转化率为97％。反应液经过滤、层析分离，浓缩结晶干燥后获得NMN纯品0.13g，NMN产品纯度99.4％。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (8)

  1. 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。
  2. 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。
  3. 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。
  4. 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。
  5. 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。
  6. 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。
  7. 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。
  8. 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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