CN115927513A - Method for preparing beta-nicotinamide mononucleotide by using biological enzyme - Google Patents
Method for preparing beta-nicotinamide mononucleotide by using biological enzyme Download PDFInfo
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Abstract
The invention relates to the technical field of biology, and discloses a method for preparing beta-nicotinamide mononucleotide by using biological enzyme. The invention takes D-ribose, nicotinamide, adenosine triphosphate ATP or adenosine diphosphate ADP or adenosine monophosphate AMP as raw materials, NMN is synthesized under the catalysis of ribokinase, phosphoribosyl pyrophosphate kinase and nicotinamide phosphoribosyl pyrophosphate kinase, and polyphosphate is introduced into a reaction system to form ATP regeneration cycle, so that the process cost is greatly reduced, and the large-scale industrial production is facilitated.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a method for producing nicotinamide mononucleotide by enzymatic catalysis, in particular to a method for preparing beta-nicotinamide mononucleotide by using biological enzyme.
Background
beta-Nicotinamide Mononucleotide (NMN) is a biochemical substance existing in biological cells, is converted into Nicotinamide adenine dinucleotide (NAD, also called coenzyme I) which is an important substance for the survival of cells after adenylylation of beta-Nicotinamide adenosine transferase, is directly involved in adenosine transfer in vivo, and the level of the Nicotinamide adenine dinucleotide in the biological cells directly influences the concentration of the NAD, plays an important role in energy generation of the biological cells and has no harm to human bodies.
Until now, nicotinamide mononucleotide has been found to have various medical and health care effects such as delaying senility, treating senile diseases such as Parkinson, regulating insulin secretion, influencing mRNA expression and the like, more applications are continuously developed, and the demand of nicotinamide mononucleotide on the market is increased along with the increase of the cognition of people on the medical and health care effects of nicotinamide mononucleotide and the wide application of nicotinamide mononucleotide as a reaction substrate in the chemical industry.
The current preparation method of NMN mainly includes the following three methods: 1. yeast fermentation method, 2. Chemical synthesis method, 3. Biological enzyme catalysis method. Among them, the chemical synthesis method has disadvantages of high cost and generation of chiral compounds; NMN produced by a yeast fermentation method contains a certain organic solvent residue; the biocatalysis method does not contain organic solvent residue, does not have chiral problem, and the prepared NMN and the same type in an organism become the most green, environment-friendly and pollution-free NMN preparation method at present. The existing biocatalysis method for preparing NMN mainly has two ways. One is the catalytic production of NMN from Nicotinamide Ribokinase (NRK) in the presence of ATP using Nicotinamide Riboside (NR) as the starting material. The NMN yield of the currently reported route is low, and the substrate raw materials are expensive; secondly, the Nicotinamide and 5-phosphoribosyl-1-pyrophosphate (PRPP) are used as substrates to prepare the NMN under the catalysis of Nicotinamide phosphoribosyltransferase (NAMPT), wherein the PRPP has high market price and limited source, so that the production cost of the biocatalysis method is high, and the application and development of the biocatalysis method are severely restricted.
There is therefore a need to develop a new method for the preparation of NMN using highly efficient biocatalytic techniques with low cost substrates.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for synthesizing beta-nicotinamide mononucleotide by multienzyme catalysis, which takes one of D-ribose, nicotinamide, adenosine triphosphate ATP, adenosine diphosphate ADP or adenosine monophosphate AMP as a raw material, prepares NMN by a one-pot method under the coupling catalysis of ribokinase RK, phosphoribosyl pyrophosphate kinase PRS and nicotinamide phosphoribosyl transferase NAMPT, and simultaneously introduces polyphosphate kinase PPK2 III and polyphosphate to realize the cyclic regeneration of ATP and further reduce the production cost.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method for preparing beta-nicotinamide mononucleotide by an enzyme method comprises the steps of taking polyphosphate, nicotinamide, D-ribose and ATP as substrates, and catalytically synthesizing beta-nicotinamide mononucleotide NMN by polyphosphate kinase, ribokinase, phosphoribosyl pyrophosphate kinase and nicotinamide phosphoribosyltransferase, wherein ATP is recycled.
In the above technical solution, further, ATP in the substrate is replaced by ADP or AMP; preferably, ADP or AMP replaces ATP in equal amounts.
In the above technical scheme, further, the ribokinase RK is derived from escherichia coli, and the amino acid sequence of ribokinase is shown in SEQ ID No.1 (e.coli source);
the phosphoribosyl pyrophosphate kinase PRS is derived from escherichia coli, and the amino acid sequence of the phosphoribosyl pyrophosphate kinase is shown as SEQ ID NO.2 (E.coli source);
the nicotinamide phosphoribosyltransferase NAMPT is derived from human, and the amino acid sequence of the nicotinamide phosphoribosyltransferase is shown as SEQ ID NO.3 (from Homo sapiens);
the polyphosphate kinase PPK2 III is derived from Thermus subsp, and the amino acid sequence of the polyphosphate kinase is shown in SEQ ID NO.4 (from Meiothermus rubber).
In the above technical scheme, further, the catalytic synthesis reaction temperature is 20-50 ℃, preferably 30-40 ℃; the reaction pH is 6.5 to 8.5, preferably 7.5 to 8.0.
In the above technical solution, further, the catalytic synthesis reaction is performed in a PBS buffer or a Tris-HCl buffer; preferably PBS buffer; the concentration of the PBS buffer solution or the Tris-HCl buffer solution is 50mM.
In the above technical solution, further, the molar ratio of the 3 substrates D-ribose, ATP, nicotinamide is 1:1-5:1-5, adding the raw materials according to the proportion to ensure that the D-ribose fully reacts, and the conversion rate of the D-ribose reaches 90-100 percent; preferably 1:1-2:1-3, the conversion rate of D-ribose reaches 100 percent; the amount of the ribokinase enzyme added was 3mg/L, the amount of phosphoribosyl pyrophosphate kinase enzyme was 0.35mg/L, the amount of nicotinamide phosphoribosyl transferase enzyme was 1.8g/L, and the amount of polyphosphate kinase enzyme was 1mg/L.
In the technical scheme, coenzyme factor Mg is further added into the reaction system 2+ And K + Coenzyme factor Mg 2+ The concentration is 5-50mM, K + The concentration is 50-150mM 2+ And K + Is one or more of magnesium chloride, magnesium sulfate, magnesium sulfite, magnesium nitrate, potassium chloride, potassium sulfate and potassium nitrate.
In the above technical solution, the polyphosphate is selected from one or more of sodium tripolyphosphate, potassium tripolyphosphate, sodium hexametaphosphate, potassium tetrametaphosphate, and sodium tetrametaphosphate, preferably sodium hexametaphosphate, and is present in excess in the system.
In the above technical solution, further, the expression host of ribokinase RK, phosphoribosyl pyrophosphate kinase PRS, nicotinamide phosphoribosyl transferase NAMPT, and polyphosphate kinase PPK2 iii is a microorganism, and the microorganism is at least one of escherichia coli, bacillus subtilis, and saccharomyces cerevisiae, preferably escherichia coli, and further preferably escherichia coli BL21 (DE 3). Recombinant expression vectors for enzymes in microorganisms are various vectors conventional in the art, including: at least one of various plasmids, cosmids, phages and viral vectors, such as pET28a (+), cosmid vectors, lambda phage vectors, preferably prokaryotic expression vector pET28a (+).
In the above technical solution, further, the specific existing forms of the ribokinase RK, phosphoribosyl pyrophosphate kinase PRS, nicotinamide phosphoribosyl transferase NAMPT, and polyphosphate kinase PPK2 iii include enzyme solution, enzyme lyophilized powder, enzyme-containing cells, immobilized enzyme and immobilized enzyme-containing cells, crude enzyme without purification, or partially purified and completely purified forms, preferably purified enzyme solution.
The reaction process of the invention is as follows: d-ribose and ATP are firstly synthesized into 5-phosphoribosyl R-5-P under the action of ribokinase RK, then PRPP is generated by the reaction of the R-phosphoribosyl R-P and ATP under the catalysis of phosphoribosyl pyrophosphate kinase PRS, and finally the beta-nicotinamide mononucleotide NMN is obtained by the reaction of the PRPP and nicotinamide NAM under the catalysis of nicotinamide phosphoribosyl transferase NAMPT.
In the presence of polyphosphate, ADP and AMP are catalyzed by PPK2 III to regenerate ATP, so that the ATP can be recycled. Or ADP and AMP generated in the process of catalytically synthesizing NMN are used as substrates, ATP is generated through catalysis of polyphosphate kinase, and a specific reaction formula of the regeneration cycle of the ATP is shown in figure 1.
Compared with the prior art, the invention has the following beneficial effects:
the invention effectively reduces the production cost, uses D-ribose with lower cost as a raw material to replace PRPP with high price by using a multi-enzyme one-pot method for co-catalysis, simultaneously has simple and convenient operation compared with the traditional step feeding method, can continuously carry out multi-step reaction on reactants, shortens the reaction time, improves the reaction efficiency, converts ADP and AMP generated in the reaction process into ATP by introducing polyphosphate and PPK2 III enzyme, realizes the cyclic regeneration of the ATP, can directly use the ADP and AMP with lower price as substrates for production, also realizes the cyclic regeneration of the ATP, reduces the cost of the whole process again, and is more favorable for large-scale industrial production.
Drawings
FIG. 1 is a process flow diagram of the preparation of beta-nicotinamide mononucleotide NMN by a multienzyme one-pot method of the invention;
FIG. 2 is an SDS-PAGE picture of a protein expressed by ribokinase BL21 (DE 3) of the present invention;
FIG. 3 is an SDS-PAGE picture of phosphoribosyl pyrophosphate kinase BL21 (DE 3) expression protein of the present invention;
FIG. 4 is a SDS-PAGE pattern of the expression protein of nicotinamide phosphoribosyltransferase BL21 (DE 3) of the present invention;
FIG. 5 is an SDS-PAGE picture of the polyphosphate kinase BL21 (DE 3) expression protein of the present invention.
Detailed Description
The invention is further illustrated, but is not in any way limited, by the following specific examples.
Example 1 construction of enzyme engineering strains
The amino acid sequences (the sequences are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in sequence) of ribokinase, phosphoribosyl pyrophosphate kinase, nicotinamide phosphoribosyl transferase and polyphosphate kinase are sent to a gene synthesis company for codon optimization of escherichia coli, encoding genes are artificially synthesized and cloned between NdeI and XhoI enzyme cutting sites of a prokaryotic expression vector pET28a (+). The recombinant expression vector containing the 4 enzyme genes is introduced into Escherichia coli BL21 (DE 3) by a chemical transformation method to obtain corresponding enzyme gene expression engineering strains. The engineering strain is fermented by adopting a conventional fermentation culture medium LB, and OD is cultured under the conditions of 37 ℃ and 220rpm 600 Reaching 0.6-0.8, inducing expression with IPTG (isopropyl-beta-D-thiogalactoside), collecting thallus, crushing, and detecting the expression of target protein (see figure 2, figure 3, figure 4, and figure 5). Centrifuging the successfully expressed bacterial liquid, collecting thalli, cracking cells, separating protein from other components, purifying the target protein through affinity chromatography and gel filtration chromatography to finally obtain the target proteinAnd (3) enzyme solution.
EXAMPLE 2 purified enzyme one-pot preparation of NMN (without ATP cycle)
50mM PBS buffer solution, 0.5mM D-ribose, 1mM ATP, 3mM nicotinamide NAM, and 10mM MgCl were added to the reaction system 2 And 125mM KCl, adjusting the pH to 7.5-8.0, then adding 3mg/L ribokinase RK, 0.35mg/L phosphoribosyl pyrophosphate kinase PRS and 1.8g/L nicotinamide phosphoribosyl transferase NAMPT, controlling the reaction temperature at 37 ℃, maintaining the pH at 7.5-8.0, and reacting for 1h to obtain a crude product solution of beta-nicotinamide mononucleotide, thus obtaining 0.03mM NMN with the yield of 10.1mg/L.
EXAMPLE 3 one-pot preparation of NMN with purified enzyme (ATP cycling Using ATP as initial substrate)
50mM PBS buffer solution, 0.5mM D-ribose, 1mM ATP, 3mM nicotinamide NAM, and 10mM MgCl were added to the reaction system 2 And 125mM KCl,10mM sodium hexametaphosphate, adjusting the pH to 7.5-8.0, then adding 3mg/L ribokinase RK, 0.35mg/L phosphoribosyl pyrophosphate kinase PRS, 1.8g/L nicotinamide phosphoribosyl transferase NAMPT and 1mg/L polyphosphate kinase PPK2 III, controlling the reaction temperature at 37 ℃, maintaining the pH at 7.5-8.0, and obtaining a crude product solution of beta-nicotinamide mononucleotide after reacting for 1h to obtain 0.09mM NMN with the yield of 29.9mg/L.
EXAMPLE 4 purification of enzyme preparation of NMN by one-pot method (ATP cycling Using ADP as initial substrate)
50mM PBS buffer solution, 0.5mM D-ribose, 1mM ADP, 3mM nicotinamide NAM, and 10mM MgCl were added to the reaction system 2 And 125mM KCl,10mM sodium hexametaphosphate, adjusting the pH to 7.5-8.0, then adding 3mg/L ribokinase RK, 0.35mg/L phosphoribosyl pyrophosphate kinase PRS, 1.8g/L nicotinamide phosphoribosyl transferase NAMPT and 1mg/L polyphosphate kinase PPK2 III, controlling the reaction temperature at 37 ℃, maintaining the pH at 7.5-8.0, obtaining a crude product solution of beta-nicotinamide mononucleotide after reacting for 1h, obtaining 0.049mM NMN, and obtaining the yield of 16.4mg/L.
EXAMPLE 5 one-pot preparation of NMN with purified enzyme (ATP cycling with AMP as initial substrate)
50mM Tris-HCl buffer solution and 0.5mM D-Ribose, 1mM AMP, 3mM nicotinamide NAM, 10mM MgCl 2 And 125mM KCl,10mM sodium hexametaphosphate, adjusting the pH to 7.5-8.0, then adding 3mg/L ribokinase RK, 0.35mg/L phosphoribosyl pyrophosphate kinase PRS, 1.8g/L nicotinamide phosphoribosyl transferase NAMPT and 1mg/L polyphosphate kinase PPK2 III, controlling the reaction temperature at 37 ℃, maintaining the pH at 7.5-8.0, and obtaining a crude product solution of beta-nicotinamide mononucleotide after reacting for 1h to obtain 0.052mM NMN with the yield of 17.4mg/L.
The invention provides a novel method for preparing beta-nicotinamide mononucleotide by an enzyme method, which obviously reduces the production cost. The price of PRPP used in the prior method is about 8.3 ten thousand per gram, while the price of D-ribose used in the method is about 0.5 yuan per gram, the price of ATP is about 3.2 ten thousand per gram, after ADP and AMP are used for replacing ATP, the production cost is further reduced, the price of ADP is about 107 yuan per gram, and the price of AMP is about 19 yuan per gram.
It will be apparent to those skilled in the art that many changes and modifications can be made, or equivalents employed, to the presently disclosed embodiments without departing from the intended scope of the invention. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention shall still fall within the protection scope of the technical solution of the present invention, unless the contents of the technical solution of the present invention are departed.
Claims (10)
1. A method for preparing beta-nicotinamide mononucleotide by an enzymatic method is characterized by comprising the following steps: the method takes polyphosphate, nicotinamide, D-ribose and ATP as substrates, takes polyphosphate kinase, ribokinase, phosphoribosyl pyrophosphate kinase and nicotinamide phosphoribosyl transferase as catalysts to synthesize beta-nicotinamide mononucleotide, and simultaneously regenerates ATP circularly.
2. The method of claim 1, wherein the enzymatic method comprises the steps of: (ii) ATP in the substrate is replaced with ADP or AMP; preferably, ADP or AMP replaces ATP in equal amounts.
3. The method according to claim 1 or 2,
the ribokinase is derived from escherichia coli, and the amino acid sequence of the ribokinase is shown in SEQ ID No. 1;
the phosphoribosyl pyrophosphate kinase is derived from escherichia coli, and the amino acid sequence of the phosphoribosyl pyrophosphate kinase is shown in SEQ ID No. 2;
the nicotinamide phosphoribosyltransferase is from human, and the amino acid sequence of the nicotinamide phosphoribosyltransferase is shown in SEQ ID NO. 3;
the polyphosphate kinase is derived from Thermus subsp, and the amino acid sequence of the polyphosphate kinase is shown in SEQ ID No. 4.
4. The process according to claim 1, characterized in that the catalytic synthesis reaction temperature is 20-50 ℃, preferably 30-40 ℃; the reaction pH is 6.5 to 8.5, preferably 7.5 to 8.0.
5. The method of claim 1, wherein: the catalytic synthesis reaction is carried out in a PBS buffer solution or a Tris-HCl buffer solution; preferably a PBS buffer; the buffer concentration was 50mM.
6. The method of claim 1, wherein: the molar ratio of the 3 substrates D-ribose, ATP and nicotinamide is 1:1-5:1-5, preferably 1:1-2:1-3; the amount of the ribokinase enzyme added was 3mg/L, the amount of phosphoribosyl pyrophosphate kinase enzyme was 0.35mg/L, the amount of nicotinamide phosphoribosyl transferase enzyme was 1.8g/L, and the amount of polyphosphate kinase enzyme was 1mg/L.
7. According toThe method of claim 1, wherein: adding coenzyme factor Mg into the reaction system 2+ And K + Coenzyme factor Mg 2+ The concentration is 5-50mM, K + The concentration is 50-150mM 2+ And K + Is one or more of magnesium chloride, magnesium sulfate, magnesium sulfite, magnesium nitrate, potassium chloride, potassium sulfate and potassium nitrate.
8. The method of claim 1, wherein: the polyphosphate is selected from one or more of sodium tripolyphosphate, potassium tripolyphosphate, sodium hexametaphosphate, potassium tetrametaphosphate and sodium tetrametaphosphate, preferably sodium hexametaphosphate, and exists in excess in the system.
9. The method of claim 1, wherein: the expression hosts of the ribokinase RK, the phosphoribosyl pyrophosphate kinase PRS, the nicotinamide phosphoribosyl transferase NAMPT and the polyphosphate kinase PPK2 III are microorganisms, the microorganisms are at least one of escherichia coli, bacillus subtilis and saccharomyces cerevisiae, the escherichia coli is preferred, and the escherichia coli BL21 (DE 3) is further preferred.
10. The method of claim 1, wherein: the specific existing forms of the ribokinase RK, the phosphoribosyl pyrophosphate kinase PRS, the nicotinamide phosphoribosyl transferase NAMPT and the polyphosphate kinase PPK2 III comprise enzyme liquid, enzyme freeze-dried powder, enzyme-containing cells, immobilized enzymes and immobilized enzyme-containing cells, unpurified crude enzyme, or partially purified and completely purified forms, preferably purified enzyme liquid.
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| CN110373398A (en) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | A kind of niacinamide ribokinase mutant and its application |
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| CN110373397A (en) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | A kind of Nampt mutant and its application |
| CN110373398A (en) * | 2019-08-06 | 2019-10-25 | 江苏诚信药业有限公司 | A kind of niacinamide ribokinase mutant and its application |
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