CN111187759A - Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by using enzyme method - Google Patents
Enzyme composition for preparing nicotinamide mononucleotide and method for preparing nicotinamide mononucleotide by using enzyme method Download PDFInfo
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Abstract
The invention relates to the technical field of biological pharmacy and biochemical engineering, in particular to an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing nicotinamide mononucleotide by an enzyme method. The enzyme composition consists of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase. The three enzymes are reasonably combined to efficiently catalyze and prepare the nicotinamide mononucleotide. The enzyme composition disclosed by the invention can be recycled, is low in cost, and is energy-saving and environment-friendly. The method for preparing nicotinamide mononucleotide by the enzymatic method takes adenosine as a substrate, and can prepare nicotinamide mononucleotide safely and reliably at low cost by adding the enzyme composition, so that the cost of the existing route is reduced, the nicotinamide mononucleotide is suitable for large-scale production, and the use of nicotinamide mononucleotide in the fields of biocatalysis and medicines is guaranteed.
Description
Technical Field
The invention relates to the technical field of biological pharmacy and biochemical engineering, in particular to an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing nicotinamide mononucleotide by an enzyme method.
Background
Nicotinamide Mononucleotide (NMN) is the substrate for the synthesis of coenzyme I and has the following structural formula:
it becomes coenzyme I (NAD) upon adenylation by nicotinamide riboside adenyltransferase. The level of NMN and the activity of nicotinamide nucleotide adenosine transferase (NAMPT) in organisms directly influence the concentration of NAD, and simultaneously NMN directly participates in adenosine transfer in vivo, and is an important synthetic substrate and function regulating substance in vivo. In the aspect of treatment and application, NMN can be used for resisting aging, treating chronic diseases and the like, and meanwhile, research shows that NMN also has a regulating effect on the secretion of insulin and has an influence on the expression level of mRNA. Therefore, NMN has wide application prospect in the aspect of medical treatment and also has wide market prospect in the aspect of chemical engineering as a reaction substrate.
NMN generally uses ion exchange resin to purify, because it is very similar to many kinds of analogues such as NAD electric charge and polarity, there is great difficulty in separating and purifying, can't remove the analogue impurity completely, so the product purity that the ion exchange method obtains is only about 60%, the yield is only 40%, the production efficiency is low, is not suitable for the large-scale production. Accordingly, the prior art is yet to be improved and developed.
Compared with the prior art, the method for producing NMN by biological enzyme catalysis is more efficient, the catalytic enzyme can be recycled in the whole reaction process, the cost is low, and the method is energy-saving and environment-friendly. At present, two methods are used for producing NMN, one method is to take nicotinamide, AMP and ATP as substrates and generate NMN through the catalytic reaction of adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase; the other one is that nicotinamide, ribose and ATP are used as substrates, and NMN is generated through the catalytic reaction of ribokinase and nicotinamide ribophosphotransferase. Compared with adenosine as a raw material, AMP (adenosine monophosphate) as a production raw material is more expensive. Therefore, the method using AMP as a raw material for production is rarely applied to practical production. At present, no report of NMN production by taking adenosine as a substrate exists in the prior art.
Disclosure of Invention
In view of the above, the present invention provides an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing nicotinamide mononucleotide by an enzymatic method. The method takes adenosine as a raw material, and the three enzymes of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase are reasonably combined to efficiently catalyze and prepare the nicotinamide mononucleotide with low cost.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides an enzyme composition for preparing nicotinamide mononucleotide, which consists of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase.
Preferably, the enzyme activity ratio of the adenosine kinase, the adenine phosphoribosyl transferase and the nicotinamide phosphoribosyl transferase is (2-3): (1-2): (0.5 to 1).
Preferably, the ratio of the enzymatic activities of the adenosine kinase, the adenine phosphoribosyltransferase and the nicotinamide phosphoribosyltransferase is 2.5: (1-2): (0.5 to 1).
In the specific embodiment provided by the invention, the enzyme activity ratio of adenosine kinase, adenine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase is 2.5: 1: 0.5.
in the specific embodiment provided by the invention, the enzyme activity ratio of adenosine kinase, adenine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase is 2.5: 2: 0.5.
in the specific embodiment provided by the invention, the enzyme activity ratio of adenosine kinase, adenine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase is 2.5: 1: 1.
preferably, the adenosine kinase, adenine phosphoribosyltransferase or nicotinamide phosphoribosyltransferase is present as a free enzyme solution, immobilized enzyme or immobilized recombinant cell.
The invention also provides a method for preparing nicotinamide mononucleotide by an enzymatic method, which comprises the following steps: adenosine, nicotinamide, ATP, MgCl2Mixing the phosphate buffer solution and the enzyme composition according to any one of claims 1 to 4 to obtain an enzyme reaction solution, and catalyzing the reaction to obtain nicotinamide mononucleotide.
Preferably, the enzymeIn the reaction solution, the concentration of adenosine is 18-22 g/L, the concentration of nicotinamide is 12-15 g/L, the concentration of ATP is 56-64 g/L, and MgCl is added2The concentration of the adenosine kinase is 9-12 g/L, and the enzyme activity of the adenosine kinase is 2 multiplied by 105～3×105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105～2×105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105～1×105U。
Preferably, the concentration of adenosine in the liquid enzyme reaction mixture is 20g/L, the concentration of nicotinamide is 13.7g/L, the concentration of ATP is 60g/L, and MgCl is added2The concentration of (A) is 10.3g/L, and the enzymatic activity of adenosine kinase is 2.5X 105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105～2×105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105～1×105U。
In the specific embodiment provided by the present invention, the concentration of adenosine in the liquid enzyme reaction mixture is 20g/L, the concentration of nicotinamide is 13.7g/L, the concentration of ATP is 60g/L, and MgCl is added2The concentration of (A) is 10.3g/L, and the enzymatic activity of adenosine kinase is 2.5X 105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105U。
In the specific embodiment provided by the present invention, the concentration of adenosine in the liquid enzyme reaction mixture is 20g/L, the concentration of nicotinamide is 13.7g/L, the concentration of ATP is 60g/L, and MgCl is added2The concentration of (A) is 10.3g/L, and the enzymatic activity of adenosine kinase is 2.5X 105The enzyme activity of U, adenine phosphoribosyl transferase is 2X 105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105U。
In the specific embodiment provided by the present invention, the concentration of adenosine in the liquid enzyme reaction mixture is 20g/L, the concentration of nicotinamide is 13.7g/L, the concentration of ATP is 60g/L, and MgCl is added2The concentration of (A) is 10.3g/L, and the enzymatic activity of adenosine kinase is 2.5X 105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 1 × 105U。
Preferably, the pH of the phosphoric acid buffer solution is 7.0.
Preferably, the conditions for the catalytic reaction are: the reaction temperature is 30-35 ℃, and the reaction time is 6-10 hours.
Preferably, the conditions of the catalytic reaction are: the reaction temperature was 33 ℃ and the reaction time was 8 hours.
The invention provides an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing nicotinamide mononucleotide by an enzyme method. The enzyme composition consists of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase. The invention has the following technical effects:
the invention reasonably combines three enzymes to efficiently catalyze and prepare the nicotinamide mononucleotide. The enzyme composition disclosed by the invention can be recycled, is low in cost, and is energy-saving and environment-friendly. The method for preparing nicotinamide mononucleotide by the enzymatic method takes adenosine as a substrate, and can prepare nicotinamide mononucleotide safely and reliably at low cost by adding the enzyme composition for producing nicotinamide mononucleotide, so that the cost of the existing route is reduced, the nicotinamide mononucleotide is suitable for large-scale production, and the use of nicotinamide mononucleotide in the fields of biocatalysis and medicines is guaranteed.
The invention adds adenosine kinase on the basis of the original NMN production enzyme system, and the enzyme can catalyze adenosine to generate AMP. The price of adenosine is only 30 percent of that of AMP, the adenosine is low in price and wide in source, and the cost is saved for industrial production of NMN.
Detailed Description
The invention discloses an enzyme composition for preparing nicotinamide mononucleotide and a method for preparing nicotinamide mononucleotide by using the enzyme method, and a person skilled in the art can use the content to reference the text and appropriately improve process parameters to realize the preparation. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The invention provides an enzyme composition, which consists of adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase.
The enzyme composition provided by the invention can be used for enzymatic catalytic synthesis of NMN by reasonably combining three enzymes, namely Adenosine Kinase (AK), adenine phosphoribosyl transferase (APRT) and nicotinamide phosphoribosyl transferase (NmPRT).
In some embodiments, the enzyme composition has a mass ratio of adenosine kinase, adenine phosphoribosyltransferase, nicotinamide phosphoribosyltransferase of 1:1: 1.
In some embodiments, the ratio of adenosine kinase, adenine phosphoribosyltransferase, nicotinamide phosphoribosyltransferase is 1:2: 1.
In some embodiments, the ratio of adenosine kinase, adenine phosphoribosyltransferase, nicotinamide phosphoribosyltransferase is 1:1: 2.
In some embodiments, the ratio of adenosine kinase, adenine phosphoribosyltransferase, nicotinamide phosphoribosyltransferase is 2:1: 1.
The adenosine kinase, adenine phosphoribosyl transferase and nicotinamide phosphoribosyl transferase in the enzyme composition are related enzyme preparations obtained by amplifying target fragments through PCR (polymerase chain reaction) by using a gene engineering technology, connecting the target fragments with a carrier, transferring the target fragments into a host bacterium, and inducing protein expression. The enzyme preparation can be crude enzyme liquid or dry powder, and can also be immobilized enzyme or immobilized recombinant cells after being immobilized.
The immobilized enzyme preparation or immobilized recombinant cells can be separated and recovered by centrifugation or filtration. The recovered immobilized enzyme or immobilized recombinant cell can be reused for enzymatic reaction.
The invention also provides a method for preparing nicotinamide mononucleotide by using the enzyme composition.
A method for preparing nicotinamide mononucleotide by enzyme method comprises adding adenosine, nicotinamide, ATP, MgCl into pH7.0 phosphate buffer solution2·6H2O, mixing evenly, adding the enzyme composition,catalyzing to obtain nicotinamide mononucleotide.
The method takes adenosine as a substrate, and carries out enzymatic reaction in a biological catalysis system by adding the enzyme composition for producing the nicotinamide mononucleotide, so that the nicotinamide mononucleotide can be prepared safely and reliably at low cost. The specific reaction formula is as follows:
in some embodiments, in the method for preparing nicotinamide mononucleotide by using enzymatic method of the present invention, the final concentration of adenosine is 18 g/L-22 g/L, the final concentration of nicotinamide is 12 g/L-15 g/L, the final concentration of ATP is 56 g/L-64 g/L, and MgCl is used2·6H2The final concentration of O is 20 g/L-24 g/L. In some embodiments, the final concentration of adenosine is 20g/L, nicotinamide is 13.7g/L, ATP is 60g/L, MgCl2·6H2The final concentration of O was 22 g/L.
Preferably, in the method for preparing nicotinamide mononucleotide by using the enzymatic method, the catalytic reaction is carried out for 6-10 hours under the conditions of pH7.0 and reaction temperature of 30-35 ℃.
The reaction solution separated after the catalytic reaction can be used for preparing a finished product of the nicotinamide mononucleotide by ion exchange chromatography, concentration, crystallization, drying and the like.
Adenosine Kinase (AK) enzyme activity determination method: the reaction system is as follows: 1mL of 0.1M MgAC21mL of 1M Gly-NaOH (pH 9.0), 2mL of 0.1% bromothymol blue, 48mg of ATP and 53mg of adenosine, and ultrapure water was added to a total volume of 20mL to obtain a reaction mixture having the following concentrations of the respective components: 5mM MgAC2(magnesium acetate), 50mM Gly-NaOH (glycine-sodium hydroxide), 0.01% bromothymol blue, 4.7mM ATP, and 10mM adenosine. The absorbance at 614nm was adjusted to about 2.2. + -. 0.3 with 0.5M NaOH to bring the pH of the reaction mixture to about 7.4. + -. 0.3, and the reaction mixture was ready for use. AK (3.68mg/mL) was diluted with Tris-buffer (20mM TrisHCl, 500mM NaCl, 5% glycerol, pH8.0) in a gradient, and 5. mu.L of the diluted BmADK was added to 995. mu.L of the reaction mixture using a 1cm light pathThe absorbance of the reaction system at 614nm over 180s was recorded on a plastic cuvette on a DU800 nucleic acid/protein analyzer (Beckman, USA). The reaction rate was defined as the slope of the absorbance change line at 614nm 10s before the reaction.
The enzyme activity determination method of Adenine Phosphoribosyltransferase (APRT) comprises the following steps: the enzyme reaction system and the reaction conditions are as follows: 100mM Tris-HCl (pH7.0), 20mM MgCl21mM AMP and 1mM nicotinamide, reacting for 15min at 30 ℃, boiling for 5min to inactivate enzyme, centrifuging, and passing the supernatant through a membrane. Detecting the content of adenine by HPLC, specifically C18 HPLC chromatographic column, 250mm × 4.6 mm; mobile phase: aqueous triethylamine phosphate solution (with a phosphoric acid content of 0.6% (v/v), pH adjusted to 6.6 with triethylamine): methanol 90: 10; an ultraviolet detector with wavelength of 254nm, column temperature of 30 ℃, flow rate of 1mL/min and sample amount of 5 muL.
The enzyme activity determination method of nicotinamide phosphoribosyltransferase (NmPRT) comprises the following steps of carrying out an enzyme reaction system and reaction conditions including 100mM Tris-HCl (pH7.0), 1mM ATP, 1mM nicotinamide, 1mM 5-phosphorus- α -D-ribose 1-diphosphate, reacting for 15min at 30 ℃, inactivating enzyme with hydrochloric acid, centrifuging, enabling supernate to pass through a membrane, detecting the NMN content by adopting HPLC (high performance liquid chromatography), specifically a C18 HPLC chromatographic column with the thickness of 250mM multiplied by 4.6mM, and carrying out a mobile phase including 0.1% TFA, methanol, an ultraviolet detector, the wavelength of 260nm, the column temperature of 25 ℃, the flow rate of 0.8mL/min and the sample injection amount of 5 mu L.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available. Wherein the formula of the seed culture medium is 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl10 g. The formula of the fermentation medium is 10g/L of peptone, 5g/L of yeast powder, 8g/L of NaCl, 8g g/L of glycerol, 1.2g/L of monopotassium phosphate, 1.8g/L of monopotassium phosphate and 1g/L of magnesium sulfate.
The invention is further illustrated by the following examples:
example 1 preparation of enzyme for NMN production
3 pairs of amplification primers were designed based on the sequences of 3 enzyme genes. Extracting genomic DNA of an Escherichia coli (Escherichia coli) strain, using the genomic DNA as a template, amplifying an AK fragment by PCR, and connecting the AK fragment to a pET28a vector (purchased from Novagene); extracting Escherichia coli (Escherichia coli) strain genome DNA, using the Escherichia coli strain genome DNA as a template, carrying out PCR amplification on an APRT fragment, and connecting the APRT fragment to a pET28a vector (purchased from Novagene); extracting genomic DNA of Haemophilus ducreyi (Haemophilus ducreyi) (purchased from GmbH, Czeri, Shanghai), performing PCR amplification on an NmPRT gene fragment by taking the genomic DNA as a template, and connecting the NmPRT gene fragment to a pET28a vector (purchased from Novagene); after the 3 gene fragments are successfully connected and sequenced correctly, the gene fragments are respectively transferred into E.coli BL21(DE3) strain (Shanghai Diego Biotechnology Co., Ltd.).
Wherein the sequence of Adenosine Kinase (AK) is: atgcgtatcattctgcttggcgctccgggcgcggggaaagggactcaggctcagttcatcatggagaaatatggtattccgcaaatctccactggcgatatgctgcgtgctgcggtcaaatctggctccgagctgggtaaacaagcaaaagacattatggatgctggcaaactggtcaccgacgaactggtgatcgcgctggttaaagagcgcattgctcaggaagactgccgtaatggtttcctgttggacggcttcccgcgtaccattccgcaggcagacgcgatgaaagaagcgggcatcaatgttgattacgttctggaattcgacgtaccggacgaactgatcgttgaccgtatcgtcggtcgccgcgttcacgcgccgtctggtcgtgtttatcacgttaaattcaatccgccgaaagtagaaggtaaagacgacgttaccggtgaagaactgactacccgtaaagacgatcaggaagaaaccgtacgtaaacgtctggttgaataccatcagatgacagcaccgctgatcggctactactccaaagaagcggaagcgggtaacaccaaatacgcgaaagttgacggcaccaagccggttgctgaagttcgcgctgatctggaaaaaatcctcggc, respectively; the sequence of the amplification primer is AK-F5 '-3': agtcgcatcatatgcgtatcattctgctt, and the restriction is marked as enzyme cutting site NdeI; AK-R3' -5:. gcgcgccgatagaattcgccgaggattttttccThe restriction site EcoRI is underlined.
The sequence of Adenine Phosphoribosyltransferase (APRT) is: atgaccgcgactgcacagcagcttgagtatctcaaaaatagcatcaaaagcattcaggactacccaaaacccggcattcttttccgcgatgtcaccagcttactggaagacccgaaagcttacgctctcagcatcgacttgctggttgagcgttacaaaaatgcgggcattaccaaagttgtcggcaccgaagcgcgtggcttcttgtttggcgctccggtagctctgggtctgggcgttggctttgtaccggtccgtaaaccgggcaaactgccgcgtgaaaccatcagtgaaacttacgacctggaatacggcaccgatcagctggagatccacgttgatgccatcaaaccgggcgacaaagttctggtggtggacgacctgctggcaaccggcggcactatcgaagcgaccgttaaactgatccgtcgtctgggtggtgaagtggctgacgctgcgttcattatcaacctgttcgatctcggcggcgaacagcgtctcgaaaaacagggcattaccagctacagccttgtcccgttcccgggccattaa, respectively; the sequence of the amplification primer is APRT-F:5 '-3': agtcgcatcatatgaccgcgactgcacag, and drawing a line as an enzyme cutting site NdeI; APRT-R3 '-5': gcgcgccgatagaattcttaatggcccgggaacggg, the restriction site EcoRI is underlined.
The sequence of nicotinamide phosphoribosyltransferase (NmPRT) is atggataacctattaaattatagtagtcgtgctagtgctataccatcattattatgcgatttttacaaaacatctcatcgaataatgtatcccgaatgttcacaaattatttatagtacatttacacctcgtagcaatgaacaagcgccttatttaacacaagttgtgtcatttggttttcaagcctttatcattaaatatttaattcattattttaatgataactttttttctcgagataaacatgatgttgtgactgaatactctgcatttattgagaaaaccttacagttagaggatacgggtgaacacattgcaaaattacatgagttgggttatttgcctatccggattaaagctattcctgaaggaaaaacggtggcaattaaagttccggtgatgacgattgaaaatacgcattctgatttcttttggcttactaactatttagaaacattaattaatgtatcactttggcagccgatgacttctgcctcgattgcttttgcttatcggacagcattaattaaatttgctaatgaaacttgtgataatcaagaacatgtgccatttcaatcgcatgatttttcaatgcgtggtatgagttctttagaatccgcagaaacttcaggtgctggccatttaacttcttttttaggtacagacactattcctgcactctcttttgttgaagcgtattatggttcaagcagtctaattggcacgtctatacccgcttctgagcattcagtaatgagttcacatggtgtcgatgaattatcaacatttcgttatttaatggcaaaatttccgcataatatgttgtcaattgtgtcagatactacagacttttggcataacattaccgttaatttgccgttattaaagcaagaaattatagcaaggccagaaaatgcccgtttagtcattcgtccagatagcggtaacttttttgcgattatttgtggtgatccaaccgctgatactgagcatgaacgtaaaggactcattgaatgtttatgggatatttttggtggtacagttaatcagaaaggttataaagtgatcaatccacatattggggcaatttatggtgatggcgtgacttatgaaaaaatgtttaagatcttagaaggattacaagccaaaggatttgcctcaagtaatattgtgtttggcgttggtgcacaaacctatcaacgtaatacacgtgatacgttgggctttgcgcttaaagcgacatctatcactattaatggcgaagaaaaagctattttcaaaaatcctaaaaccgatgatggttttaaaaaatcgcaaaaaggtcgtgttaaagtgctttctcgtgatacttacgttgatggtttaacttcagcggatgattttagtgatgatttattagagctgttatttgaagatggtaagttattacgccaaacagactttgatgaaattcggcaaaacttgttagttagtcgcactacgctatga; the sequence of the amplification primer is NmPRT-F5 '-3': agtcgcatcatatggataacctattaaattatag, drawing a line as an enzyme cutting site NdeI; NmPRT-R3 '-5': gcgcgccgatagaattctcatagcgtagtgcgactaactaac, the restriction site EcoRI is underlined.
Inoculating the strain into a seed culture medium under an aseptic condition, inoculating the strain into a 5L fermentation culture medium fermentation tank after the strain is cultured to a logarithmic growth phase, inoculating the strain into a 50L fermentation culture medium fermentation tank after the strain is continuously cultured to the logarithmic growth phase, adding 1mMIPTG (millipore size transfer) after the strain is cultured for 5 hours, inducing the strain at 25 ℃ for 20 hours, and centrifugally collecting the strain.
The harvested thalli are respectively subjected to ultrasonic or high-pressure homogenization and crushing, then the supernatant is centrifugally collected and purified by a nickel ion affinity column, and the high-quality purified enzyme is obtained.
Preparation of immobilized production enzymes or cells: 4.0 g of polyethylene glycol are dissolved in 45m1 of water, 6.0 g of polyvinyl alcohol (PVA) are added and the temperature is raised to 90-95 ℃ for dissolution. After the polyvinyl alcohol is completely dissolved, the temperature is reduced to 25-30 ℃, recombinant cells or purified enzyme is added, the mixture is uniformly mixed, a disposable straw is used for sucking the mixed solution, the mixed solution is injected on a plane to form a disc shape, and the disc shape is subjected to warm bath for 1 hour at the temperature of 30 ℃. The solution was then transferred to a 0.1M sodium sulfate solution for stabilization for 2-3 hours, filtered off and washed twice with sterile water and placed in phosphate buffer for further use.
Using the above-described method for measuring enzyme activity, 1mg/ml of Adenosine Kinase (AK), Adenine Phosphoribosyltransferase (APRT) and nicotinamide phosphoribosyltransferase (NmPRT) enzyme activities of about 50U, 20U and 10U, respectively, were detected, wherein 1 activity unit (U) is defined as the amount of enzyme required to completely convert 1 μm of a substrate into a product within 1 minute.
Example 2 preparation of NMN
100g of adenosine substrate, 68.5g of nicotinamide, 300g of ATP and MgCl2·6H2O110 g, added to 5L of phosphate buffer solution pH7.0, stirred well, and adjusted to pH 7.0. Adding an enzyme composition consisting of AK, APRT and NmPRT into the reaction liquid, wherein the mass ratio of AK to APRT to NmPRT in the enzyme composition is 1:1:1, the pH value is controlled to be 7.0 during the reaction, the reaction temperature is controlled to be 33 ℃, after oscillation reaction is carried out for 8 hours, the NMN production amount in the reaction supernatant is 19.67g/L through HPLC detection, the purity is 55%, and the adenosine conversion rate is 96%.
HPLC detection conditions: octadecylsilane chemically bonded silica was used as a filler, mobile phase A was 0.1% TFA, mobile phase B was methanol, detection wavelength was 260nm, column temperature was 25 ℃, and elution procedure was as shown in Table 1.
TABLE 1 elution procedure
| Time (min) | Mobile phase A (%) | Mobile phase B (%) | Flow rate (ml/min) |
| 0.01 | 100 | 0 | 0.8 |
| 3.00 | 100 | 0 | 0.8 |
| 5.30 | 80 | 20 | 0.8 |
| 6.60 | 80 | 20 | 1.0 |
| 8.60 | 80 | 20 | 1.2 |
| 9.0 | 100 | 0 | 1.2 |
| 12.0 | 100 | 0 | Stop |
The supernatant collected by filtration is subjected to ion exchange chromatography, concentration, crystallization and drying by macroporous strongly basic anion exchange resin to obtain 98.38g of finished NMN with the purity of 99.0 percent and the total yield of 82 percent.
Example 3 preparation of NMN
100g of adenosine substrate, 68.5g of nicotinamide, 300g of ATP and MgCl2·6H2O110 g, added to 5L of phosphate buffer solution pH7.0, stirred well, and adjusted to pH 7.0. Adding an enzyme composition consisting of AK, APRT and NmPRT into the reaction solution, wherein the mass ratio of AK to APRT to NmPRT in the enzyme composition is 1:2:1, the pH value is controlled to be 7.0 during the reaction, the reaction temperature is controlled to be 30-35 ℃, after oscillation reaction is carried out for 8 hours, the NMN generation amount in the reaction supernatant is 17.4g/L through HPLC detection, the purity is 50%, and the adenosine conversion rate is 92%. HPLC detection conditions were the same as in example 2.
The supernatant collected by filtration is subjected to ion exchange chromatography, concentration, crystallization and drying by macroporous strongly basic anion exchange resin to obtain a finished product NMN 87.4g, the purity is 98.8 percent, and the total yield is 76 percent.
Example 4 preparation of NMN
100g of adenosine substrate, 68.5g of nicotinamide, 300g of ATP and MgCl2·6H2O110 g, added to 5L of phosphate buffer solution pH7.0, stirred well, and adjusted to pH 7.0. Adding an enzyme composition consisting of AK, APRT and NmPRT into the reaction solution, wherein the mass ratio of AK to APRT to NmPRT in the enzyme composition is 1:1:2, the pH value is controlled to be 7.0 during the reaction, the reaction temperature is controlled to be 30-35 ℃, after oscillation reaction is carried out for 8 hours, the NMN generation amount in the reaction supernatant is 18.3/L through HPLC detection, the purity is 52%, and the adenosine conversion rate is 94%. HPLC detection conditions were the same as in example 2.
The supernatant collected by filtration is subjected to ion exchange chromatography, concentration, crystallization and drying by macroporous strongly basic anion exchange resin to obtain 91.6g of finished NMN with the purity of 98.7 percent and the total yield of 78 percent.
Example 5 preparation of NMN
100g of adenosine substrate, 68.5g of nicotinamide, 300g of ATP and MgCl2·6H2O110 g, added to 5L of phosphate buffer solution pH7.0, stirred well, and adjusted to pH 7.0. Adding an enzyme composition consisting of AK, APRT and NmPRT into the reaction solution, wherein the mass ratio of AK to APRT to NmPRT in the enzyme composition is 2:1:1, the pH value is controlled to be 7.0 during the reaction, the reaction temperature is controlled to be 30-35 ℃, after oscillation reaction is carried out for 8 hours, the generation amount of NMN in the reaction supernatant is 19g/L through HPLC detection, the purity is 54%, and the adenosine conversion rate is 95%. HPLC detection conditions were the same as in example 2.
And (3) performing ion exchange chromatography, concentration, crystallization and drying on the filtered and collected supernatant through macroporous strongly basic anion exchange resin to obtain 95g of finished NMN with the purity of 98.9 percent and the total yield of 80 percent.
Example 6 detection of Activity of immobilized enzyme or immobilized cell
The immobilized enzymes or immobilized cells of the three enzymes for NMN production can be recycled, and the three immobilized enzymes for NMN production are mixed and recycled for 20 times according to the mass ratio of 1:1:1 and are subjected to tracking detection according to the known method for measuring the enzyme activity described in the prior art, and the results are shown in Table 2.
TABLE 2 Change in enzyme Activity by cycling reaction or storage at 4 ℃ of the immobilized enzyme
| Number of times of use of immobilized enzyme/storage time at 4 deg.C | Enzyme activity U/mg |
| Enzyme activity of immobilized enzyme | 20 |
| The immobilized enzyme is used for 5 times | 18.2 |
| The immobilized enzyme is used for 10 times | 17.5 |
| The immobilized enzyme is used for 15 times | 16.3 |
| The immobilized enzyme is used for 20 times | 15.1 |
| Unused storage for 15 days | 17.8 |
| Unused storage for 30 days | 15.6 |
The results show that the enzymatic activity of the three immobilized enzymes for NMN production gradually decreased with the increase in the number of cycles. The reaction was cycled 20 times and enzyme activity was only reduced by about 25%. The enzymatic activity of the three immobilized enzymes for NMN production was also reduced by only about 20-25% at a storage time of more than one month at 4 ℃. The enzyme activity is reduced by 20-25% after the mixture is circularly reacted for 20 times according to the mass ratio of 1:2:1, 1:1:2 and 2:1: 1.
In addition, the activity of the immobilized cells of the three production enzymes of NMN decreased only 20-25% with increasing cycle number or prolonged storage time at 4 ℃.
Comparative example 1
Takes nicotinamide, ribose and ATP as substrates, and generates NMN through the catalytic reaction of ribokinase and nicotinamide ribose phosphotransferase, and the yield is about 65-70%.
The yield of NMN in the invention is 75-82%, and the yield is improved.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. An enzyme composition for preparing nicotinamide mononucleotide, which is characterized by comprising adenosine kinase, adenine phosphoribosyltransferase and nicotinamide phosphoribosyltransferase.
2. The enzyme composition according to claim 1, wherein the enzyme activity ratio of the adenosine kinase, the adenine phosphoribosyltransferase and the nicotinamide phosphoribosyltransferase is (2-3): (1-2): (0.5 to 1).
3. The enzyme composition according to claim 1, wherein the enzyme activity ratio of the adenosine kinase, the adenine phosphoribosyltransferase and the nicotinamide phosphoribosyltransferase is 2.5: (1-2): (0.5 to 1).
4. The enzyme composition according to claim 1, wherein the adenosine kinase, the adenine phosphoribosyltransferase or the nicotinamide phosphoribosyltransferase is present as a free enzyme solution, an immobilized enzyme or an immobilized recombinant cell.
5. A method for preparing nicotinamide mononucleotide by an enzymatic method is characterized by comprising the following steps: adenosine, nicotinamide, ATP, MgCl2Mixing the enzyme composition of any one of claims 1 to 4 with a phosphate buffer solution to obtain an enzyme reaction mixture, and catalyzing the reaction to obtain nicotinamide mononucleotide.
6. The method according to claim 5, wherein the concentration of adenosine in the liquid enzyme reaction mixture is 18 to 22g/L, the concentration of nicotinamide is 12 to 15g/L, and the concentration of ATP is 56～64g/L，MgCl2The concentration of the adenosine kinase is 9-12 g/L, and the enzyme activity of the adenosine kinase is 2 multiplied by 105～3×105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105～2×105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105～1×105U。
7. The method according to claim 6, wherein the concentration of adenosine in the enzyme reaction mixture is 20g/L, the concentration of nicotinamide is 13.7g/L, the concentration of ATP is 60g/L, and MgCl is added2The concentration of (A) is 10.3g/L, and the enzymatic activity of adenosine kinase is 2.5X 105The enzyme activity of U, adenine phosphoribosyl transferase is 1 × 105～2×105The enzyme activity of U, nicotinamide phosphoribosyltransferase is 0.5X 105～1×105U。
8. The method of claim 5, wherein the phosphate buffer solution has a pH of 7.0.
9. The method of claim 5, the conditions of the catalytic reaction being: the reaction temperature is 30-35 ℃, and the reaction time is 6-10 hours.
10. The process according to any one of claims 5 to 9, the conditions of the catalytic reaction being: the reaction temperature was 33 ℃ and the reaction time was 8 hours.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111647635A (en) * | 2020-06-19 | 2020-09-11 | 理星（天津）生物科技有限公司 | Method for synthesizing beta-nicotinamide mononucleotide and intermediate thereof |
| CN112359082A (en) * | 2020-11-13 | 2021-02-12 | 南京工业大学 | Preparation method of nicotinamide mononucleotide |
| CN112795606A (en) * | 2021-04-14 | 2021-05-14 | 深圳瑞德林生物技术有限公司 | Enzymatic synthesis method of beta-nicotinamide mononucleotide |
| CN112961890A (en) * | 2021-02-05 | 2021-06-15 | 深圳希吉亚生物技术有限公司 | Enzymatic synthesis method of nicotinamide mononucleotide |
| CN113358776A (en) * | 2021-06-01 | 2021-09-07 | 连云港杰瑞药业有限公司 | Method for simultaneously detecting multiple related substances in NMN (N-dimethylformamide) by HPLC (high performance liquid chromatography) |
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| CN113755415A (en) * | 2020-06-04 | 2021-12-07 | 苏州华赛生物工程技术有限公司 | Novel recombinant microorganism with NMN synthetic pathway and production method thereof |
| CN114672530A (en) * | 2022-04-22 | 2022-06-28 | 江苏信佑生物有限公司 | Method for preparing beta-nicotinamide mononucleotide |
| WO2022217694A1 (en) * | 2021-04-13 | 2022-10-20 | 百瑞全球有限公司 | METHOD FOR PREPARING β-NICOTINAMIDE MONONUCLEOTIDE, AND ENZYME COMPOSITION AND APPLICATION THEREOF |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191170A (en) * | 2016-08-09 | 2016-12-07 | 深圳市古特新生生物科技有限公司 | A kind of enzyme process prepares the method for adenosine triphosphate |
| CN110195089A (en) * | 2019-06-13 | 2019-09-03 | 叁爻生物科技(上海)有限公司 | Nicotinamide mononucleotide and preparation method thereof |
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
-
2020
- 2020-01-19 CN CN202010058700.1A patent/CN111187759A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106191170A (en) * | 2016-08-09 | 2016-12-07 | 深圳市古特新生生物科技有限公司 | A kind of enzyme process prepares the method for adenosine triphosphate |
| CN110195089A (en) * | 2019-06-13 | 2019-09-03 | 叁爻生物科技(上海)有限公司 | Nicotinamide mononucleotide and preparation method thereof |
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
Non-Patent Citations (3)
| Title |
|---|
| PANKIEWICZ,KW等: "Nicotinamide Adenine Dinucleotide Based Therapeutics, Update", 《CURRENT MEDICINAL CHEMISTRY》 * |
| UMMARINO,S等: "Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay", 《FOOD CHEMISTRY》 * |
| 赵娟等: "烟酰胺单核苷酸的研究及应用进展", 《食品科技》 * |
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| CN113755415B (en) * | 2020-06-04 | 2024-03-29 | 苏州华赛生物工程技术有限公司 | Novel recombinant microorganism with NMN synthesis path and production method thereof |
| CN111647635B (en) * | 2020-06-19 | 2021-09-21 | 理星（天津）生物科技有限公司 | Method for synthesizing beta-nicotinamide mononucleotide and intermediate thereof |
| CN111647635A (en) * | 2020-06-19 | 2020-09-11 | 理星（天津）生物科技有限公司 | Method for synthesizing beta-nicotinamide mononucleotide and intermediate thereof |
| CN112359082A (en) * | 2020-11-13 | 2021-02-12 | 南京工业大学 | Preparation method of nicotinamide mononucleotide |
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