CN117143940A - Method for synthesizing beta-nicotinamide mononucleotide by using adenine phosphoribosyl transferase - Google Patents
Method for synthesizing beta-nicotinamide mononucleotide by using adenine phosphoribosyl transferase Download PDFInfo
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- PQGCEDQWHSBAJP-TXICZTDVSA-N 5-O-phosphono-alpha-D-ribofuranosyl diphosphate Chemical compound O[C@H]1[C@@H](O)[C@@H](O[P@](O)(=O)OP(O)(O)=O)O[C@@H]1COP(O)(O)=O PQGCEDQWHSBAJP-TXICZTDVSA-N 0.000 claims abstract description 42
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
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- C12N9/10—Transferases (2.)
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- C12N9/1077—Pentosyltransferases (2.4.2)
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- C12Y204/00—Glycosyltransferases (2.4)
- C12Y204/02—Pentosyltransferases (2.4.2)
- C12Y204/02007—Adenine phosphoribosyltransferase (2.4.2.7)
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Abstract
The invention discloses a method for synthesizing beta-nicotinamide mononucleotide by using adenine phosphoribosyl transferase, which takes nicotinamide and 5-phosphoribosyl-1-pyrophosphate as substrates, and the nicotinamide and the 5-phosphoribosyl-1-pyrophosphate are condensed by an adenine phosphoribosyl transferase catalytic system to generate the beta-nicotinamide mononucleotide, wherein the catalytic system consists of adenine phosphoribosyl transferase and a PRPP synthesis system. The method for synthesizing nicotinamide mononucleotide by using adenine 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 adenine phosphoribosyl transferase, which takes adenine phosphoribosyl transferase as a catalyst to stereoselectively catalyze condensation of nicotinamide and 5-phosphoribosyl-1-pyrophosphoric acid to obtain beta-nicotinamide mononucleotide.
Background
Nicotinamide mononucleotide (Nicotinamide mononucleotide, NMN), which belongs to the vitamin B group derivative, is a naturally occurring bioactive nucleotide, is a precursor of nicotinamide adenine dinucleotide (Nicotinamide adenine dinucleotide, NAD, also known as coenzyme i), and is an important intermediate metabolite in cells. NMN has two isomers, α and β, wherein the β isomer is the active form of NMN (hereinafter β -NMN is abbreviated as NMN). NAD is difficult to enter cells directly, NMN is well tolerated as a direct precursor to NAD synthesis, and recently NMN has been known for its potential to resist Aging and extend life (Tubota K.the first human clinical study for NMN has started in Japan. NPJ Aging Mech Dis.2016; 2:16021). In terms of safety, more and more clinical trials related to NMN have been approved (e.g., NCT03151239, U M I N0000 30 60 9, U M I N00002 1 30 9, and UMIN000025739, etc.). Phase II clinical trials of Igarashi et al showed that aged men continued to take NMN for 6 or 12 weeks without any adverse effects (Igarashi M, nakagawa-Nagahama Y, miura M, et al, chronic nicotinamide mononucleotide supplementation elevates blood nicotinamide adenine dinucleotide levels and alters muscle function in healthy older men, NPJ aging 2022;8 (1): 5.). NMN is therefore the preferred supplement to NAD. However, natural NMN is extremely low in the content of organisms and cannot be obtained by separation and extraction. At present, the traditional NMN preparation method is prepared by a chemical synthesis way, but the cost for preparing high-purity NMN is high due to the fact that the stereoselectivity is difficult to control, the yield is low, and the product taking the NMN as a main active ingredient is quite expensive. Compared with chemical synthesis, the biological enzyme method for synthesizing NMN has the advantages of high efficiency and environmental protection, no organic solvent residue and no chiral problem. 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.
Adenine phosphoribosyl transferase (Adenine phosphoribosyl transferase, APRT), belonging to the family of 6-oxopurine phosphoribosyl transferase (PRTase), has the biological function of catalyzing adenine and 5-phosphoribosyl-1-pyrophosphate (PRPP) to condense and generate adenine nucleoside monophosphate (AMP), and is a key enzyme of adenine salvage synthesis pathway.
Disclosure of Invention
The invention aims to provide a method for synthesizing beta-nicotinamide mononucleotide by using adenine phosphoribosyl transferase, which is a method for obtaining NMN by condensing Nicotinamide (NAM) and 5-phosphoribosyl-1-pyrophosphate (PRPP) by using adenine phosphoribosyl transferase stereoselectivity, and is an expansion application of adenine 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 adenine 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 adenine phosphoribosyl transferase and a PRPP synthesis system.
The method comprises the following specific steps: takes NAM and PRPP as raw materials and Mg 2+ As cofactor, NMN is obtained by condensation under the catalysis of adenine 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: ribose kinase and phosphoribosyl pyrophosphokinase as key enzymes of the system, ATP and D-ribose as substrates, mg 2+ PRPP synthesis system as cofactor.
The enzyme catalysis system comprises adenine phosphoribosyl transferase, ribose kinase, phosphoribosyl pyrophosphatase and magnesium chloride. The enzyme catalytic systemThe adenine phosphoribosyl transferase, ribose kinase and phosphoribosyl pyrophosphatase are all free pure enzymes after purification. Adding ATP, D-ribose, NAM and PRPP into the enzyme reaction system, and controlling the pH and temperature under the condition of Mg 2+ As cofactors, ATP phosphorylates D-ribose to 5-phosphoribosyl under the catalysis of ribokinase, and further phosphorylates 5-phosphoribosyl to PRPP under the catalysis of phosphoribosyl pyrophosphatase, and finally adenine phosphoribosyl transferase condenses NAM with PRPP to produce NMN, with the following reaction formula:
specifically, the adenine phosphoribosyl transferase nucleotide sequence is derived from GenBank, the number is M14040.1, and belongs to Escherichia coli (Escherichia coli (strain K12)), and the adenine phosphoribosyl transferase is obtained by total gene synthesis of biotechnology company as shown in EcAPRT-DNA (SEQ. No. 1) in a sequence table after coding is optimized.
Specifically, the amino acid sequence of the adenine phosphoribosyl transferase is shown as EcAPRT-AA (SEQ. No.2) in a sequence table.
As known to those skilled in the art, the nucleotide sequence of the adenine phosphoribosyl transferase gene of the present invention may be any other nucleotide sequence encoding the amino acid sequence shown as EcAPRT-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 EcAPRT-DNA is within the scope of the present invention as long as it has homology of 90% or more with nucleotides.
Any amino acid sequence shown in EcAPRT-AA is deleted, inserted or substituted by one or more amino acids and has NMN synthesis activity, and still belongs to the protection scope of the invention.
For adenine phosphoribosyl transferase, any other source of isozymes having NMN synthesizing activity falls within the scope of the present invention.
Specifically, the ribokinase sequence is derived from GenBank, accession No.: 948260, after codon optimization, is E.coli (strain K12)) ribose kinase, as shown in RBKS-DNA (SEQ. No. 3) in the sequence Listing, and is obtained by total gene synthesis of biotechnology company.
Specifically, the amino acid sequence of the ribokinase is shown as RBKS-AA (SEQ. No. 4) in the sequence table.
Specifically, the phosphoribosyl pyrophosphate kinase nucleotide sequence is derived from GenBank, and the code is: 885993 is Mycobacterium tuberculosis (Mycobacterium tuberculosis) phosphoribosyl pyrophosphate kinase, and is obtained by total gene synthesis of biotechnology company as shown by MtPRS-DNA (SEQ. No. 5) in a sequence table after codon optimization.
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 adenine phosphoribosyl transferase is 0.5-5.0mg/ml.
In the catalytic system, the addition amount of the substrate NAM is 0.5-30mM; the addition amount of PRPP is 0.75-45mM; cofactor Mg 2+ The addition amount of (C) 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 25-40 ℃ and the reaction time is 3-20h; 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 adenine phosphoribosyl transferase as a biocatalyst, which has not been reported at present; the adenine phosphoribosyl transferase has the characteristics of small molecular weight, easy purification, capability of achieving 15 mg/liter of culture medium of the heterologous expression quantity of escherichia coli, capability of obtaining free enzyme with purity of more than 95% by one-step purification through nickel affinity chromatography, reaction at the pH value close to neutral and normal temperature, no need of participation of transition metal and organic solvent, high catalytic reaction rate, high heterologous expression quantity, easy purification and the like, and meanwhile, the method has the advantages of mild reaction condition, environmental friendliness and the like, and has good industrial application development prospect.
Drawings
FIG. 1 is a SDS-PAGE of purified adenine 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 6.0h of reaction in the catalytic system of adenine phosphoribosyl transferase.
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 Ara Ding Huaxue reagent Inc., shanghai Seiyaku Biotechnology Inc., national pharmaceutical Condition chemical Co., ltd.
EXAMPLE 1 expression of adenine phosphoribosyl transferase
Coding Escherichia coli (strain K12) adenine phosphoribosyl transferase gene (GenBank: M14040.1) sequence, codon optimizing (sequence as SEQ ID NO. 1), synthesizing by biological engineering (Shanghai) limited company, adding into pET-28a (+) vector, constructing EcAPRT-pET-28a (+) plasmid, sequencing, verifying sequence, heat-shock transforming to E.coli BL21 (DE 3) competent cells, obtaining adenine phosphoribosyl transferase expression engineering bacteria, culturing in LB plate containing 50 μg/ml kanamycinPicking single colony on the culture medium, inoculating to LB liquid culture medium containing 50 μg/ml kanamycin, shaking and culturing at 37deg.C with 200rpm for 12 hr, transferring to 3L liquid LB culture medium for expansion culture, shaking and culturing at 37deg.C with 200rpm for 8 hr, and collecting culture medium with optical density OD 600 When the temperature reaches 0.6, the temperature is reduced to 23 ℃, IPTG solution with the final concentration of 0.5mM is added for induction expression for 16h, the culture solution is centrifuged at 8000rpm for 10min, the supernatant culture solution is discarded, and the thalli are preserved at-20 ℃ for standby.
EXAMPLE 2 expression of ribokinase
The coding Escherichia coli (strain K12) ribose kinase gene (GenBank: 948260) sequence is optimized by codon (sequence is shown as SEQ ID NO. 3), after being fully synthesized by biological engineering (Shanghai) limited company, the sequence is connected with pET-28a (+) vector to construct RBKS-pET-28a (+) plasmid, after sequencing and verifying the sequence, the sequence is transformed into E.coli BL21 (DE 3) competent cells by heat shock, adenine phosphoribosyl transferase expression engineering bacteria are obtained, single colony is selected from LB plate medium containing 50 mug/ml kanamycin, inoculated into LB liquid medium containing 50 mug/ml kanamycin, after shaking culture for 12 hours at 37 ℃, the culture medium is transferred into 1.5L liquid LB medium for expansion culture, after shaking culture for 8 hours at 200rpm of shaking table at 37 ℃, when the optical density OD of the culture medium is obtained 600 When the temperature reaches 0.6, the temperature is reduced to 16 ℃, IPTG solution with the final concentration of 0.2mM is added for induction expression for 16h, the culture solution is centrifuged at 8000rpm for 10min, the supernatant culture solution is discarded, and the thalli are preserved at-20 ℃ for standby.
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 (the sequence is shown as SEQ ID NO. 5), is fully synthesized by a biological engineering (Shanghai) limited company and is connected with a pET-28a (+) vector to construct an MtPRS-pET-28a (+) plasmid, after sequencing and verifying the sequence, the plasmid is transformed into E.coli BL21 (DE 3) competent cells by heat shock to obtain adenine phosphoribosyl transferase expression engineering bacteria, a single colony is picked from LB plate medium containing 50 mu g/ml kanamycin, inoculated to LB liquid medium containing 50 mu g/ml kanamycin, and based on the following stepsShaking culture at 37deg.C and 200rpm for 12 hr, transferring to 1.5L liquid LB culture medium, performing amplification culture, shaking culture at 37deg.C and 200rpm for 8 hr, and collecting culture solution with optical density OD 600 When the temperature reaches 0.6, the temperature is reduced to 16 ℃, IPTG solution with the final concentration of 0.4mM is added for induction expression for 16h, the culture solution is centrifuged at 8000rpm for 10min, the supernatant culture solution is discarded, and the thalli are preserved at-20 ℃ for standby.
EXAMPLE 4 purification of adenine phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphate kinase
Adenine phosphoribosyl transferase expression engineering bacteria or ribose kinase, phosphoribosyl pyrophosphorokinase bacteria 3g are 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 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 ℃, the lysate (50 mM imidazole, 50mM Tris-HCl,500mM NaCl,10% glycerol and 1% Tween-20 pH 8.0) is used for eluting and removing unadsorbed proteins, finally elution buffer (500 mM imidazole, 50mM Tris-HCl,500mM NaCl,10% glycerol and 1% Tween-20 pH 8.0) is used for eluting and collecting target proteins, and 5L of Kpi buffer (50 mM KH) 2 PO 4 ，50mM K 2 HPO 4 pH 7.4) to dialyze the target protein, remove salts and imidazole, SDS-PAGE analysis results show that the purified yields of adenine phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphatase can reach 10 mg/liter of culture medium, 20 mg/liter of culture medium and 8 mg/liter of culture medium respectively, and the purity is more than 95% (figure 1).
EXAMPLE 3 Synthesis of NMN by adenine phosphoribosyl transferase
The adenine phosphoribosyl transferase, ribokinase and phosphoribosyl pyrophosphatase pure enzyme obtained in example 2 were added to the reaction system in an amount of 1.0mg/ml, 50mM pH 7.4Kpi was used as a buffer, NAM in a final concentration of 0.8mM and P in a final concentration of 0.8mM were added, respectivelyRPP, 1.0mM ATP, 0.8mM D-ribose, 10mM MgCl 2 After 6.0h of reaction at 37℃with constant temperature shaking (666 rpm), the reaction was terminated by adding an equal volume of methanol, and after 15min centrifugation at 14000rpm, the supernatant was taken and fed into a High Performance Liquid Chromatography (HPLC) to analyze the amounts of substrate and product. The HPLC analysis method comprises the following steps: shimadzu high performance liquid chromatography LC-20AT; chromatographic column extension-C18.6 x 250mm; 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 NMN catalyzed by the adenine phosphoribosyl transferase by using NAM and NMN standard substance concentration curves. The conversion rate can reach 50.4% and the yield is 45.1% 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 DMSO (containing 20% acetophenone), 10. Mu.l 2M KOH, 25. Mu.l reaction supernatant were mixed, and after 2min in ice bath, 45. Mu.l 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 xanthine-guanine phosphoribosyl transferase catalyzed NMN is calculated from the NMN standard concentration curve. The yield can reach 40.1% 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 using adenine 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 adenine phosphoribosyl transferase and a PRPP synthesis system.
2. The method according to claim 1, characterized by the following steps: adopts nicotinamide and 5-phosphoribosyl-1-pyrophosphoric acid as raw materials, usesMg 2+ Condensing a substrate to obtain beta-nicotinamide mononucleotide under the catalysis of adenine phosphoribosyl transferase in an enzyme catalysis system as a cofactor, 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 according to claim 1 or 2, wherein the adenine phosphoribosyl transferase is derived from Escherichia coli (strain K12), the nucleotide sequence encoding the adenine phosphoribosyl transferase is shown in SEQ No.1, and the amino acid sequence is shown in SEQ No. 2.
4. The method according to claim 1 or 2, wherein the PRPP synthesis system is: ribose kinase and phosphoribosyl pyrophosphokinase as key enzymes of the system, ATP and D-ribose as substrates, mg 2+ As cofactor. By Mg 2+ As cofactor, ATP phosphorylates D-ribose to 5-phosphoribosyl under the catalysis of ribokinase, and further phosphorylates 5-phosphoribosyl to PRPP under the catalysis of phosphoribosyl pyrophosphatase, and the PRPP is taken as a substrate of adenine phosphoribosyl transferase and condensed with NAM to generate NMN. The reaction formula is as follows:
。
5. the method of claim 1 or 2, wherein the enzyme catalytic system comprises adenine phosphoribosyl transferase, ribokinase, phosphoribosyl pyrophosphate kinase, substrate and cofactor Mg 2+ Adenine phosphoribosyl transferase, ribokinase and phosphate in the enzyme catalytic systemThe ribose pyrophosphatase is purified to be free pure enzyme.
6. The method according to claim 5, wherein the ribokinase is derived from a ribokinase of E.coli (Escherichia coli (strain K12)).
7. The method of claim 5, wherein the phosphoribosyl-pyrophosphate kinase is derived from a phosphoribosyl-pyrophosphate kinase of mycobacterium tuberculosis (Mycobacterium tuberculosis).
8. The method according to claim 1 or 2, wherein the amount of adenine phosphoribosyl transferase added is 0.5-5.0mg/ml, the amount of ribokinase added is 0.05-3.0mg/ml, and the amount of phosphoribosyl pyrophosphate kinase added is 0.1-6.0mg/ml in the enzyme-catalyzed system.
9. The method according to claim 1 or 2, wherein the substrate nicotinamide is added in an amount of 0.5-30mM and the 5-ribose-1-pyrophosphate is added in an amount of 0.75-45mM in the enzyme catalytic system; cofactor Mg 2+ The addition amount of (C) 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. The reaction temperature is 25-40 ℃ and the reaction time is 3-20h.
10. A method according to claim 1 or 2, characterized in that the pH of the reaction is controlled to 6-9.
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