CN117925754A - Method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase - Google Patents
Method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase Download PDFInfo
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- phosphoribosyl transferase
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- 238000000034 method Methods 0.000 title claims abstract description 24
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- 230000002194 synthesizing effect Effects 0.000 title claims abstract description 21
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase, which takes nicotinamide and 5-phosphoribosyl-1-pyrophosphoric acid as substrates, and the nicotinamide mononucleotide is generated by condensing the nicotinamide and the 5-phosphoribosyl-1-pyrophosphoric acid by a uracil phosphoribosyl transferase catalytic system, wherein the catalytic system consists of uracil phosphoribosyl transferase and a PRPP synthesis system. The method for synthesizing nicotinamide mononucleotide by using uracil phosphoribosyl transferase as a catalyst has the advantages of simple reaction steps, mild reaction conditions, high catalytic rate, environment friendliness, simple enzyme expression and purification and the like, and has good application and development prospects in the field of industrial synthesis of beta-nicotinamide mononucleotide and related products thereof.
Description
Technical Field
The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a method for synthesizing beta-nicotinamide mononucleotide by using uracil phosphoribosyl transferase, which takes uracil phosphoribosyl transferase as a catalyst to stereoselectively catalyze condensation of nicotinamide and 5-phosphoribosyl-1-pyrophosphoric acid to obtain the beta-nicotinamide mononucleotide.
Background
Beta-nicotinamide mononucleotide (beta-nicotinamide mononucleotide, NMN) is a precursor of nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NAD, also known as coenzyme I), an important intermediate metabolite in cells. NAD is difficult to enter cells directly, NMN is a direct precursor to NAD synthesis, is well tolerated, and can prevent age-related physiological decline (Mills KF,Yoshida S,Stein LR,et al.Long-Term Administration of Nicotinamide Mononucleotide Mitigates Age-Associated Physiological Decline in Mice.Cell Metab.2016,24,795-806), and is therefore the first material of choice for NAD supplements. In addition, NMN has been shown to be effective in treating high fat diet-induced type 2 diabetes by reversing mitochondrial dysfunction associated with aging (Haigis MC,Mostoslavsky R,Haigis KM,et al.SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic beta cells.Cell.2006,126,941-954). but natural NMN is extremely low in organism and cannot be obtained by isolation. At present, the traditional NMN preparation method is prepared by a chemical synthesis way, but the stereoselectivity is difficult to control, the yield is lower, the cost for preparing the high-purity NMN is higher, and the product taking the NMN as the main active ingredient is quite expensive. Compared with chemical synthesis, the biological enzyme method for synthesizing NMN has the advantages of high efficiency, environmental protection, no organic solvent residue, no chiral problem and the like. At present, nicotinamide phosphoribosyl transferase for catalyzing NMN synthesis is limited by factors such as large molecular weight, complex structure, difficult directed evolution and the like, and a novel efficient phosphoribosyl transferase for synthesizing NMN is still to be developed.
Uracil phosphoribosyl transferase (Uracil phosphoribosyltransferase, UPRT), belonging to the family of phosphoribosyl transferases (PRTase), has a biological function reported at present of catalyzing uracil to condense with 5-phosphoribosyl-1-pyrophosphate (PRPP) to generate uracil nucleoside monophosphate (UMP), which is a key enzyme of pyrimidine salvage synthesis pathway.
Disclosure of Invention
The invention aims to provide a method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase, which is a novel phosphoribosyl transferase for synthesizing NMN, is a method for obtaining NMN by uracil phosphoribosyl transferase stereoselectivity catalyzing Nicotinamide (NAM) and 5-phosphoribosyl-1-pyrophosphate (PRPP) condensation, and is an expansion application of uracil phosphoribosyl transferase substrate spectrum. The method has the advantages of easy preparation of the enzyme catalyst, strong operability, simple reaction steps, high conversion efficiency and the like.
The invention provides a method for synthesizing NMN by using uracil phosphoribosyl transferase, which takes NAM and PRPP as substrates and obtains NMN through catalysis of an enzyme catalysis system, wherein the enzyme catalysis system consists of uracil phosphoribosyl transferase and a PRPP synthesis system.
The method comprises the following specific steps: NAM and PRPP are used as raw materials, mg 2+ is used as a cofactor, and NMN is obtained through condensation under the catalysis of uracil phosphoribosyl transferase in an enzyme catalysis system, and the reaction formula is as follows:
in the reaction process, NAM and PRPP are used as substrates, but the PRPP is expensive and easy to decompose, and the PRPP is synthesized by the PRPP synthesis system, so that the reaction cost can be obviously reduced.
In the invention, the PRPP synthesis system is as follows: PRPP synthesis system with ribose kinase and phosphoribosyl pyrophosphatase as key enzymes, ATP and D-ribose as substrate and Mg 2+ as cofactor.
The enzyme catalysis system comprises uracil phosphoribosyl transferase, ribose kinase, phosphoribosyl pyrophosphatase and magnesium chloride. The uracil phosphoribosyl transferase, ribose kinase and phosphoribosyl pyrophosphatase in the enzyme catalysis system are all free pure enzymes after purification. Adding ATP, D-ribose, NAM and PRPP into the enzyme reaction system, taking Mg 2+ as a cofactor under the condition of controlling pH and temperature, and phosphorylating the D-ribose into 5-phosphoribosyl under the catalysis of ribokinase by the ATP, further phosphorylating the 5-phosphoribosyl into the PRPP under the catalysis of ribokinase, and finally condensing NAM and PRPP by uracil phosphoribosyl transferase to generate NMN, wherein the reaction formula is as follows:
Specifically, uracil phosphoribosyl transferase is derived from escherichia coli (ESCHERICHIA COLI), a nucleotide sequence for encoding the ESCHERICHIA COLI phosphoribosyl transferase is derived from GenBank, the number is X57104.1, and after codon optimization, the nucleotide sequence is shown as EcUPRT-DNA (SEQ.No.1) in a sequence table.
Specifically, the amino acid sequence of uracil phosphoribosyl transferase is shown as EcUPRT-AA (SEQ. No.2) in a sequence table.
As known to those skilled in the art, the nucleotide sequence of the uracil phosphoribosyl transferase gene of the present invention may also be any other nucleotide sequence encoding the amino acid sequence shown as EcUPRT- -AA (SEQ. No. 2) in the sequence Listing.
Any nucleotide sequence obtained by substitution, certainty or insertion treatment of one or more nucleotides with respect to the nucleotide sequence shown in EcUPRT- -DNA is within the scope of the present invention as long as it has homology of 90% or more with the nucleotides.
Any pair EcUPRT-AA in which one or more amino acids have been deleted, inserted or substituted and which has NMN synthesis activity still falls within the scope of the present invention.
For uracil phosphoribosyl transferase, any other source of isozymes having NMN synthesizing activity is within the scope of the invention.
Specifically, the ribokinase sequence is derived from escherichia coli (ESCHERICHIA COLI (strain K12)), and the nucleotide sequence encoding the ribokinase is derived from GenBank, accession No.: 948260, as shown in the sequence Listing as RBKS-DNA (SEQ. No. 3), is obtained by total gene synthesis.
Specifically, the amino acid sequence of the ribokinase is shown as RBKS-AA (SEQ. No. 4) in the sequence Listing.
Specifically, the phosphoribosyl pyrophosphate kinase sequence is derived from mycobacterium tuberculosis (Mycobacterium tuberculosis), and the nucleotide sequence for encoding the phosphoribosyl pyrophosphate kinase is derived from GenBank, and the code is: 885993, shown as MtPRS-DNA (SEQ. No.5) in the sequence Listing, is obtained by total gene synthesis.
Specifically, the amino acid sequence of the phosphoribosyl pyrophosphate kinase is shown as MtPRS-AA (SEQ. No.6) in a sequence table.
Preferably, in the catalytic system, the addition amount of the ribose kinase is 0.05-3.0mg/mL; the addition amount of phosphoribosyl pyrophosphokinase is 0.1-6.0mg/mL; the addition amount of uracil phosphoribosyl transferase is 0.1-5.0mg/mL.
In the catalytic system, the addition amount of the substrate NAM is 1-60mM; PRPP is added in an amount of 1.5-90mM; the addition amount of the cofactor Mg 2+ is 0.1-20mM. The addition amount of the substrate ATP in the PRPP synthesis system is 1.0-30mM; the addition amount of D-ribose is 0.5-10mM.
Preferably, in the enzyme catalytic system, the reaction temperature is 25-37 ℃ and the reaction time is 2-20h; more preferably, the temperature is 30-37 ℃ and the time is 4-14h.
Preferably, the pH value of the reaction is controlled to be 6 to 9, the pH is controlled to be lowered by sodium hydroxide, and the pH is controlled to be raised by formic acid.
The beneficial effects of the invention are mainly as follows: provides a method for obtaining NMN by catalyzing condensation of NAM and PRPP by taking uracil phosphoribosyl transferase as a biocatalyst, which has not been reported at present; the uracil phosphoribosyl transferase has high catalytic reaction speed, can reach reaction balance within 4 hours, has small molecular weight, is easy to purify, can obtain free enzyme with purity of more than 95% by one-step purification of a culture medium with the heterologous expression amount of escherichia coli of 10mg/L, and can simultaneously react at the pH value close to neutral and normal temperature without the participation of transition metal and organic solvent, thereby having the characteristics of high catalytic reaction rate, high heterologous expression amount, easy purification and the like, simultaneously having the advantages of mild reaction conditions, environmental friendliness and the like and having good industrial application development prospect.
Drawings
FIG. 1 is a SDS-PAGE of purified uracil phosphoribosyl transferase.
Fig. 2 is a NAM blank high performance liquid chromatogram.
FIG. 3 is a high performance liquid chromatogram of the reaction solution after reaction for 4 hours in the uracil phosphoribosyl transferase catalytic system.
Detailed Description
The invention is further described below with reference to the drawings and specific examples. It should be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
The experimental methods in the present invention are conventional methods unless otherwise specified.
The plasmid extraction kit is purchased from Tiangen biochemical technology (Beijing) limited company; e.coli DH 5. Alpha. E.coli BL21 (DE 3) and the like are purchased from the division of biological engineering (Shanghai); pre-stained protein Maker was purchased from New Saimei Biotechnology Inc. of Suzhou.
Common reagents used in the present invention include substrates purchased from Aba Ding Huaxue reagent Inc., shanghai Seiyaku Biotechnology Inc., national pharmaceutical Condition chemical Co., ltd.
EXAMPLE 1 expression of uracil phosphoribosyl transferase
The coding E.coli (ESCHERICHIA COLI) uracil phosphoribosyl transferase gene (GenBank: X57104.1) sequence is optimized by a codon (the sequence is shown as SEQ ID NO. 1), is fully synthesized by a biological engineering (Shanghai) limited company, is connected into a pET-28a (+) carrier, is constructed into a EcUPRT-pET-28a (+) plasmid, is subjected to sequencing verification sequence, is subjected to heat shock conversion to E.coli BL21 (DE 3) competent cells, so as to obtain uracil phosphoribosyl transferase expression engineering bacteria, a single colony is picked from LB flat plate medium containing 50 mug/mL kanamycin, is inoculated into LB liquid medium containing 50 mug/mL kanamycin, is subjected to shaking culture for 12 hours at 37 ℃, is transferred into 3L liquid LB medium for expansion culture, is subjected to shaking culture for 8 hours at 200rpm at 37 ℃, when the optical density OD 600 of the culture solution reaches 0.6 ℃, is reduced to 16 ℃, is added into IPTG solution with the final concentration of 0.5mM for inducing expression for 16 hours, the culture solution is subjected to centrifugation for 8000rpm for 10 minutes, the supernatant is discarded, and the culture solution is kept at 20 ℃ for standby.
EXAMPLE 2 expression of ribokinase
The coding E.coli (ESCHERICHIA COLI (strain K12)) ribose kinase gene (GenBank: 948260) sequence is subjected to codon optimization (the sequence is shown as SEQ ID NO. 3), is fully synthesized by a biological engineering (Shanghai) limited company, is connected into a pET-28a (+) vector, is constructed into a RBKS-pET-28a (+) plasmid, is subjected to sequencing verification sequence, is subjected to heat shock conversion into E.coli BL21 (DE 3) competent cells, so as to obtain uracil phosphoribosyl transferase expression engineering bacteria, a single colony is picked from LB plate medium containing 50 mug/mL kanamycin, is inoculated into LB liquid medium containing 50 mug/mL kanamycin, is subjected to shaking culture for 12 hours at 37 ℃, is transferred into 1.5L liquid LB medium for expansion culture at 200rpm, is subjected to shaking culture for 8 hours at 37 ℃, when the optical density OD 600 of the culture solution reaches 0.6, is reduced to 16 ℃, is added into IPTG solution with the final concentration of 0.2mM for inducing expression for 16 hours, the culture solution is subjected to centrifugation at 8000rpm, the supernatant is removed for 10 ℃ for 20 minutes, and the strain is preserved for later use.
EXAMPLE 3 expression of phosphoribosyl pyrophosphate kinase
The coding mycobacterium tuberculosis (Mycobacterium tuberculosis) phosphoribosyl pyrophosphate kinase gene (GenBank: 885993) sequence is subjected to codon optimization (sequence is shown as SEQ ID NO. 5), is fully synthesized by a biological engineering (Shanghai) limited company, is connected into a pET-28a (+) carrier, is constructed into MtPRS-pET-28a (+) plasmid, is subjected to sequencing verification sequence, is transformed into E.coli BL21 (DE 3) competent cells through heat shock, uracil phosphoribosyl transferase expression engineering bacteria are obtained, a single colony is picked from LB plate medium containing 50 mug/mL kanamycin, inoculated into LB liquid medium containing 50 mug/mL kanamycin, subjected to shaking culture at 200rpm at 37 ℃, transferred into 1.5L liquid LB medium for expansion culture, subjected to shaking culture at 200rpm at 37 ℃ for 8 hours, when the optical density OD 600 of the culture solution reaches 0.6 ℃, is reduced to 16 ℃, IPTG solution with the final concentration of 0.4mM is added for inducing expression for 16 hours, the culture solution is centrifuged at 8000rpm for 10 minutes, the supernatant is discarded, and the culture solution is kept at 20 ℃ for later use.
EXAMPLE 4 purification of uracil phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphate kinase
Uracil phosphoribosyl transferase expression engineering bacteria or ribokinase, phosphoribosyl pyrophosphatase thallus 3g is resuspended in 20mL lysate (10 mM imidazole, 50mM Tris-HCl,500mM NaCl,10% glycerol, 1% Tween-20 pH 8.0). Shaking, adding lysozyme (1 mg/mL), ice-bathing for 40min, ultrasonic crushing for 3 times, 3 min/time, and centrifuging at 14000rpm for 15min at intervals of 15min each time to obtain supernatant as crude enzyme solution. Ni-IDA protein purification magnetic beads are used as purification materials, 5mL of 10% magnetic bead suspension is used for a single time, 10mL of lysate is used for balancing the Ni-IDA magnetic beads, crude enzyme solution is added after magnetic separation, mixed incubation is carried out for 1h at 4 ℃, unadsorbed proteins are eluted by lysate (50 mM imidazole, 50mM Tris-HCl,500mM NaCl,10% glycerol and 1% Tween-20 pH 8.0), finally target proteins are eluted and collected by eluting buffer (500 mM imidazole, 50mM Tris-HCl,500mM NaCl,10% glycerol and 1% Tween-20 pH 8.0), target proteins are dialyzed by 5L of Kpi buffer (50 mM KH 2PO4,50mM K2HPO4 and pH 7.4), and the analysis result of SDS-PAGE shows that the purified yields of uracil phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphatase can reach 10mg/L medium, 20mg/L medium and 8mg/L medium respectively, and the purity is more than 95% (figure 1).
EXAMPLE 5 uracil phosphoribosyl transferase Synthesis of NMN
Uracil phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphatase pure enzyme obtained in example 4 are added into a reaction system according to the adding amount of 0.8mg/mL, 50mM pH 7.4Kpi is taken as buffer, NAM with the final concentration of 0.8mM, PRPP with the final concentration of 0.8mM, ATP with the final concentration of 1.0mM, D-ribose with the final concentration of 0.8mM and MgCl 2 with the final concentration of 10mM are respectively added for reaction for 4 hours, constant temperature oscillation (666 rpm) at 37 ℃ is carried out, equal volume of methanol is added for stopping the reaction, and after the reaction solution is centrifuged for 15min, a supernatant liquid is taken to sample a high performance liquid phase (HPLC) for analyzing the substrate and the product amount. The HPLC analysis method comprises the following steps: shimadzu high performance liquid chromatography LC-20AT; chromatographic column extension-C18.6X1250 mm; column temperature is 30 ℃; the flow rate is 0.7mL/min; detection wavelength 245nm; mobile phase: 10% of water (100 mM sodium phosphate salt) and 90% of methanol. And calculating the conversion rate and the yield of the catalytic production of NMN by uracil phosphoribosyl transferase according to NAM and NMN standard substance concentration curves. The conversion rate can reach 40.2% and the yield is 38.6% through calculation. The corresponding high performance liquid chromatograms are shown in fig. 2 and 3. The fluorescence analysis method comprises the following steps: mu.L of DMSO (containing 20% acetophenone), 10. Mu.L of 2M KOH, 25. Mu.L of reaction supernatant were mixed, and after 2min of ice bath, 45. Mu.L of 88% formic acid was added and incubated at 37℃for 10min. Then, 40. Mu.L of the reacted solution was added to a 384-well blackboard, and fluorescence was detected at an excitation wavelength of 382nm and an emission wavelength of 445 nm. The yield of uracil phosphoribosyl transferase catalyzed NMN is calculated from the NMN standard concentration curve. The yield can reach 41.2% by calculation, which is consistent with the result obtained by HPLC detection.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (10)
1. A method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase is characterized in that nicotinamide and 5-phosphoribosyl-1-pyrophosphate are taken as substrates, and the beta-nicotinamide mononucleotide is obtained by catalysis of an enzyme catalysis system, wherein the enzyme catalysis system consists of uracil phosphoribosyl transferase and a PRPP synthesis system.
2. The method for synthesizing beta-nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 1, which is characterized by the following steps: taking nicotinamide and 5-phosphoribosyl-1-pyrophosphate as raw materials, taking Mg 2+ as a cofactor, and condensing a substrate under the catalysis of uracil phosphoribosyl transferase in an enzyme catalysis system to obtain beta-nicotinamide mononucleotide, wherein the reaction formula is as follows:
The PRPP is synthesized by taking the synthesis of PRPP as a precondition and taking ATP and D-ribose as substrates through a PRPP synthesis system through twice catalytic phosphorylation reactions of ribose kinase and phosphoribosyl pyrophosphate kinase.
3. The method for synthesizing beta-nicotinamide mononucleotide by using uracil phosphoribosyl transferase according to claim 1 or 2, wherein the uracil phosphoribosyl transferase is derived from escherichia coli (ESCHERICHIA COLI), the nucleotide sequence of the coding uracil phosphoribosyl transferase is shown as SEQ.No.1, and the amino acid sequence of the coding uracil phosphoribosyl transferase is shown as SEQ.No.2.
4. The method for synthesizing beta-nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 1 or 2, wherein the PRPP synthesis system is: ribose kinase and phosphoribosyl pyrophosphate kinase are used as key enzymes of a system, ATP and D-ribose are used as substrates, mg 2+ is used as cofactor, mg 2+ is used as cofactor, ATP is catalyzed by ribokinase to phosphorylate D-ribose into 5-phosphoribosyl, and then 5-phosphoribosyl is catalyzed by phosphoribosyl pyrophosphate kinase to be phosphorylated into PRPP which is used as a substrate of uracil phosphoribosyl transferase, and NMN is generated by condensation with NAM, wherein the reaction formula is as follows:
5. The method for synthesizing beta-nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 1 or 2, wherein the enzyme catalysis system comprises uracil phosphoribosyl transferase, ribokinase, phosphoribosyl pyrophosphatase, substrate and cofactor Mg 2+, and the uracil phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphatase in the enzyme catalysis system are all purified to be free pure enzyme.
6. The method for synthesizing β -nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 5, wherein the ribokinase is derived from ribokinase of E.coli (ESCHERICHIA COLI (strain K12)).
7. The method for synthesizing β -nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 5, wherein the phosphoribosyl-pyrophosphate kinase is derived from phosphoribosyl-pyrophosphate kinase of Mycobacterium tuberculosis (Mycobacterium tuberculosis).
8. The method for synthesizing beta-nicotinamide mononucleotide by using uracil phosphoribosyl transferase according to claim 1 or 2, wherein the addition amount of uracil phosphoribosyl transferase is 0.1-5.0 mg/mL, the addition amount of ribokinase is 0.05-3.0mg/mL, and the addition amount of phosphoribosyl pyrophosphatase is 0.1-6.0mg/mL in an enzyme catalysis system.
9. The method for synthesizing beta-nicotinamide mononucleotide by uracil phosphoribosyl transferase according to claim 1 or 2, wherein the addition amount of substrate nicotinamide is 1-60mM and the addition amount of 5-phosphoribosyl-1-pyrophosphate is 1.5-90mM in an enzyme catalytic system; the addition amount of the cofactor Mg 2+ is 0.1-20mM, and the addition amount of the substrate ATP in the PRPP synthesis system is 1.0-30mM; the addition amount of the D-ribose is 0.5-10mM, the reaction temperature is 25-37 ℃ and the reaction time is 2-20h.
10. A method for synthesizing β -nicotinamide mononucleotide using uracil phosphoribosyl transferase according to claim 1 or 2, wherein the pH of the reaction is controlled to be 6-9.
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