CN116590255A - Nicotinamide ribokinase mutant and application thereof in preparation of beta-nicotinamide mononucleotide - Google Patents
Nicotinamide ribokinase mutant and application thereof in preparation of beta-nicotinamide mononucleotide Download PDFInfo
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- FZAQROFXYZPAKI-UHFFFAOYSA-N anthracene-2-sulfonyl chloride Chemical compound C1=CC=CC2=CC3=CC(S(=O)(=O)Cl)=CC=C3C=C21 FZAQROFXYZPAKI-UHFFFAOYSA-N 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims description 8
- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 title description 20
- 235000005152 nicotinamide Nutrition 0.000 title description 10
- 239000011570 nicotinamide Substances 0.000 title description 10
- 229960003966 nicotinamide Drugs 0.000 title description 10
- 101001076781 Fructilactobacillus sanfranciscensis (strain ATCC 27651 / DSM 20451 / JCM 5668 / CCUG 30143 / KCTC 3205 / NCIMB 702811 / NRRL B-3934 / L-12) Ribose-5-phosphate isomerase A Proteins 0.000 title description 5
- 102000046755 Ribokinases Human genes 0.000 title description 5
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- 229940101270 nicotinamide adenine dinucleotide (nad) Drugs 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
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- BRFMYUCUGXFMIO-UHFFFAOYSA-N phosphono dihydrogen phosphate phosphoric acid Chemical compound OP(O)(O)=O.OP(O)(=O)OP(O)(O)=O BRFMYUCUGXFMIO-UHFFFAOYSA-N 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
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- 150000003290 ribose derivatives Chemical class 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- GCLGEJMYGQKIIW-UHFFFAOYSA-H sodium hexametaphosphate Chemical compound [Na]OP1(=O)OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])OP(=O)(O[Na])O1 GCLGEJMYGQKIIW-UHFFFAOYSA-H 0.000 description 1
- 235000019982 sodium hexametaphosphate Nutrition 0.000 description 1
- 235000019830 sodium polyphosphate Nutrition 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01022—Ribosylnicotinamide kinase (2.7.1.22)
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- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
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Abstract
The invention discloses a nicotinamide riboside kinase mutant and application thereof in preparing beta-nicotinamide mononucleotide, wherein the amino acid sequence of the nicotinamide riboside kinase mutant is at least one mutation compared with the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence derived from the amino acid sequence and having at least 80% of sequence identity. The nicotinamide riboside kinase mutant starts from wild NRK, and is subjected to directed evolution to obtain the NRK mutant capable of efficiently synthesizing beta-NMN, and the mutant enzyme is used for efficiently synthesizing the beta-NMN by NR, so that the production cost is low, the period is short and the purity is high.
Description
Technical Field
The invention belongs to the technical field of biological enzyme engineering, and particularly relates to a nicotinamide riboside kinase mutant and application thereof in preparation of beta-nicotinamide mononucleotide.
Background
Beta-nicotinamide mononucleotide (beta-NMN) is a naturally occurring biologically active nucleotide. It is Nicotinamide Adenine Dinucleotide (NAD) + ) Plays a vital role in a variety of biological processes such as metabolism, aging, cell death, DNA repair, and gene expression. NAD (NAD) + The level tends to decrease in multiple organs with age, supplementing NAD + May be an effective treatment for a variety of age-related disorders. However, NAD is inhibited due to cell membrane barrier + Enter human cells and directly feed NAD + Is less absorbent. Oral beta-NMN supplements NAD + In another form, beta-NMN is transported across the cell membrane by active transport and is then efficiently converted to NAD in human cells + . Several preclinical studies have shown that β -NMN has a variety of pharmacological activities in cerebral ischemia, alzheimer's disease, diet and age induced type ii diabetes and obesity, and recently it has been found in mouse models that β -NMN has anti-aging, life-prolonging properties that make β -NMN attractive as a potential candidate therapeutic. Due to these expected effects, β -NMN has begun to be used as a nutraceutical for self-administration.
Initially, the synthesis of β -NMN used chemical methods, but chemical synthesis was inefficient and environmentally unfriendly, with the biologically inactive byproduct α -NMN. Enzyme catalysis is a better alternative method for manufacturing valuable chemicals, has the advantages of no toxicity, high efficiency, strong substrate specificity, single product, mild production conditions and the like, and has attracted wide attention in the production of beta-NMN. Currently, enzymatic catalytic synthesis of β -NMN currently has 3 alternative routes: (1) Nicotinamide Mononucleotide (NMN) and byproduct pyrophosphoric acid (PPI) are produced by catalyzing nicotinamide phosphoribosyl transferase (Nampt) with Nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) as substrates under the participation of ATP; (2) Nicotinamide Mononucleotide (NMN) and corresponding byproducts are produced by catalyzing phosphoric acid pyrophosphoric acid Synthase (PRPPs) and nicotinamide riboside transferase (Nampt) with Nicotinamide (NAM) and D-5-phosphoribosyl phosphate as substrates under the participation of ATP, and the route is the extension of the route one: PRPP is expensive, so relatively cheap D-5-phosphoribosyl phosphate is used as a substrate, and Phosphoribosyl Pyrophosphate Synthase (PRPPs) synthesizes phosphoribosyl pyrophosphate (PRPP) in the presence of ATP; (3) Nicotinamide Riboside (NR) is used as a substrate, and Nicotinamide Riboside Kinase (NRK) catalyzes to generate Nicotinamide Mononucleotide (NMN) and a byproduct ADP in the presence of ATP.
Of the three routes, route (1) requires PRPP as a phosphate donor, which is expensive and unstable; route (2) uses relatively simple and low-cost substrates such as ribose to replace synthetic PRPP, but needs a plurality of enzyme and substrate combinations and a plurality of enzymatic reaction steps, which often results in low yield of beta-NMN and high production cost. In the route (3), NR is converted into beta-NMN by only one enzyme NRK in one step, the process is short, no intermediate product is generated, so that the method has higher conversion rate and lower cost, in addition, the NR has good stability and low price, can be synthesized by a ribose derivative and a nicotinic acid derivative (or NAM) through a chemical and enzymatic method, simultaneously, NRK and polyphosphate kinase (PPK) are combined for use, ATP is recycled in the synthesis process, and the conversion rate of NMN is improved while the consumption of ATP is reduced. Therefore, the NR synthesis path has good application prospect in the synthesis of beta-NMN. However, the wild type NRK has low activity and poor conversion rate, and needs to be further solved.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a nicotinamide riboside kinase mutant and application thereof in preparing beta-nicotinamide mononucleotide, and the NRK mutant capable of efficiently synthesizing beta-NMN is obtained from wild NRK through directed evolution, and the efficient synthesis of beta-NMN by NR is realized by using mutant enzyme, so that the production cost is low, the period is short, and the purity is high.
The invention is realized by the following technical scheme:
a nicotinamide riboside kinase mutant having at least one mutation in its amino acid sequence compared to the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence derived therefrom having at least 80% sequence identity.
Further, the nicotinamide riboside kinase mutant amino acid sequence is mutated at least one of positions 24-32 and 102-111 compared to the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence derived therefrom having at least 80% sequence identity.
Further, compared with the amino acid sequence with at least 80% sequence identity derived from the amino acid sequence shown in SEQ ID No. 1, the amino acid sequence of the nicotinamide ribokinase mutant is subjected to any one of single mutation, pairwise combined mutation or triple combination mutation at the 28 th, 29 th, 103 th and 107 th positions.
Further, compared with the amino acid sequence with at least 80% sequence identity derived from the amino acid sequence shown in SEQ ID No. 1, the amino acid sequence of the nicotinamide ribokinase mutant is subjected to triple combination mutation at 28 th, 29 th and 103 th, wherein the N at 28 th is mutated into M, the A at 29 th is mutated into R, and the F at 103 th is mutated into S.
The invention discloses application of a nicotinamide riboside kinase mutant in preparation of beta-nicotinamide mononucleotide.
Further, nicotinamide ribose is used as a substrate, ATP is used as a phosphate donor, and beta-nicotinamide mononucleotide is generated by the reaction under the catalysis of nicotinamide riboside kinase mutant.
Further, the nicotinamide riboside kinase mutant is used in combination with a polyphosphate kinase.
The nicotinamide riboside kinase mutant in the invention is selected from:
A. single mutant N28X 1, a29X2, F103X 3, e 107X4;
B. two by two, mutations (N28X 1, A29X 2), (N28X 1, F103X 3) and (N28X 1, E107X 4), (A29X 2, F103X 3), (A29X 2, E107X 4) or (F103X 3, E107X 4). C. A triple junction mutation (N28X1,A 29X2,F 103X3), (N28X1,A 29X2,E 107X4) or (a 29X2,F 103X3,E 107X4);
D. or corresponding single, double or triple mutations of amino acid sequences derived from SEQ ID NO. 1 having at least 80% sequence identity;
wherein X1, X2, X3, X4 in each case independently of one another represent the mutated amino acid residue and are any other amino acid residue which differs from the amino acid residue prior to mutation.
The invention also relates to a vector for encoding the gene, which is a pET expression vector, a pCW expression vector, a pUC expression vector, a PHT expression vector, a pPIC9k, a pPIC3.5k or a pPICZA expression vector; and host cells encoding the gene vector, which are E.coli, B.subtilis or Pichia pastoris.
Polyphosphate Kinase (PPK) is derived from Corynebacterium glutamicum, cereibacter sphaeroides, deinococcus radiodurans, escherichia coli, hydrogenophilaceae bacterium, meiothermus cerbereus, mycobacterium tuberculosis, sulfurovum lithotrophicum, sinorhizobium meliloti, thermus thermophilus, or Thermosynechococcus vestitus. Nicotinamide ribokinase mutants were used in combination with polyphosphate kinase in the form of sodium polyphosphate (Na (n+2) PnO (3n+1) N > 4) is used as a substrate to regenerate ATP, so that the consumption of ATP is reduced, and the conversion rate of NMN is improved.
The beneficial effects obtained by the invention are as follows:
(1) The nicotinamide riboside kinase mutant has improved enzyme activity, improved stability in a reaction system, and higher conversion rate under the condition of high concentration substrate (more than or equal to 100 g/L), and almost catalyzes all substrates to be converted into products; after the conversion rate is improved, the subsequent purification step is convenient, the NR and the beta-NMN are almost not needed to be separated, and the purity of the obtained beta-NMN product is more than 98 percent;
(2) The beta-nicotinamide mononucleotide prepared by the nicotinamide riboside kinase mutant enzyme method can be completely reacted in an aqueous phase system without adding an organic reagent, so that the pollution is greatly reduced, the reaction efficiency is high, and the enzyme cost is reduced.
Detailed Description
The following detailed description of the preferred embodiments of the invention is provided to enable those skilled in the art to more readily understand the advantages and features of the invention.
The Nicotinamide Riboside Kinase (NRK) mutant is a mutant of wild nicotinamide riboside kinase Kluyveromyces marxianus, the amino acid sequence of the wild nicotinamide riboside kinase Kluyveromyces marxianu is shown as SEQ ID No. 1, and the nucleic acid sequence is shown as SEQ ID No. 2.
The NRK (wild type NRK or NKR mutant) is obtained by the following steps:
obtaining recombinant escherichia coli (or other microorganism) expression strain of NRK by utilizing a molecular cloning technology and a genetic engineering technology, and then fermenting the recombinant escherichia coli to prepare recombinant cells or fermentation liquor containing NRK; both the molecular weight reduction technique and the genetic engineering technique are known. The molecular cloning technique can be found in the fourth edition of the "molecular cloning Experimental guidelines" (Michael R.Green and Joseph.Sambrook works); the expression steps of constructing the recombinant strain of the invention by adopting the genetic engineering technology are as follows:
(1) After optimizing the synthetic gene codon according to the codon preference of the escherichia coli, the synthetic gene codon is connected into a pET-28a (+) vector, and both ends of the synthetic gene codon are respectively cut by using NcoI and XhoI enzyme sites.
(2) The recombinant plasmid is transformed into competent cells of escherichia coli BL21 (DE 3) to obtain recombinant escherichia coli expression strain for expressing NRK, and IPTG is utilized to induce target protein expression.
The preparation of NRK mutant by using recombinant E.coli expression strain containing NRK mutant comprises picking 2-3 recombinant E.coli BL21 (DE 3) single colony from three-region streak LB plate, inoculating into 50m1 liquid LB culture medium, shake culturing at 37deg.C (220 rpm) until 0D600 reaches 0.6-0.8, adding 0.5mm IPTG, shake culturing at 30deg.C for 3-5 hr. After the induction, the cells were collected by centrifugation (8,000 rpm,10 min), resuspended in 0.05M phosphate buffer (pH 7.0), and further disrupted by a high-pressure continuous flow cell disrupter, centrifuged (8,000 rpm,10 min), and the supernatant was collected to obtain an NRK crude enzyme solution.
The present invention will be described in more detail with reference to specific examples.
Example 1
Enzyme activity determination:
20mL of the reaction system: 100g/L Nicotinamide Riboside (NR) and 1.4 molar equivalents of ATP are dissolved in 12mL ddH 2 After O, the pH was adjusted to 7.0 with 2.0M NaOH, and then 2mL of NRK (wild-type NRK or NRK mutant) supernatant (crude enzyme solution in phosphate buffer pH 7.0) and MgCl at a final concentration of 2mM were added 2 Finally add ddH 2 O to a total volume of 20mL. The reaction is carried out in a constant temperature water bath at 30 ℃ under the magnetic stirring state. ATP was not added under the same conditions as the control experiment. The pH was maintained at 7.0 by auto-titration of 1.0M NaOH. One enzyme activity unit (U) is defined as: catalytic NR production per minuteThe amount of 1. Mu. Mol of beta-NMN enzyme is expressed as U/ml.
Table 1: wild-type NRK enzyme and NRK mutant enzyme activity
| Enzymes | Relative enzyme Activity (%) |
| Wild type | 100 |
| Single mutant N28M | 187 |
| Single mutant A29T | 112 |
| Double mutant (N28S, A29R) | 108 |
| Double mutant (N28S F103K) | 169 |
| Triple mutant (N28M, A29R, F103S) | 365 |
| Triple mutant (N28M, A29R, E107A) | 289 |
Example 2
Stability determination
NRK (wild-type NRK or NRK mutant) was placed in a phosphate buffer system at 30 ℃ and ph7.0, respectively, and the substrate concentrations employed in the enzyme activity detection system were each set for 1 hour, and the enzyme activity was detected, and the stability of NRK was verified. The results are shown in Table 2 below:
table 2: wild-type NRK enzyme and NRK mutant enzyme stability
Example 3
Biocatalysis of NRK mutants
Dissolving 150mg of substrate NR in 1.6mL of 50mM phosphate buffer solution with pH7.0, adding 50mM sodium hexametaphosphate, 5mM ATP, 50mM magnesium sulfate, NRK and PPK (the enzyme amount is 1g/L and 0.5g/L respectively) after the substrate is completely dissolved, stirring and reacting at a constant temperature of 25 ℃ under a magnetic stirrer, and performing HPLC detection after reacting for 6 hours; substrate conversion for the different NRK mutants is shown in table 3 below:
TABLE 3 conversion of wild type and different mutants to different concentrations of substrate
| Enzymes | Conversion rate |
| Wild type | 46.3 |
| Single mutant N28M | 97.8 |
| Single mutant A29T | 96.5 |
| Double mutant (N28S, A29R) | Complete conversion |
| Double mutant (N28S F103K) | Complete conversion |
| Triple mutant (N28M, A29R, F103S) | 98.7 |
| Triple mutant (N28M, A29R, E107A) | Complete conversion |
Example 4
In a 50ml reaction system, 120g/L NR, 5mM ATP and 10mM MgC1 were added in this order 2 NRK mutant crude enzyme solution (protein content 5 g/L) prepared by the triple mutant (N28M, A29R, E107A) in example 1 is evenly mixed and then placed in a water bath at 30 ℃, stirred at 300rpm for reaction for 12 hours, and after the reaction is finished, the NR conversion rate is detected by HPLC to be more than 99.91%. After ion exchange resin separation, freeze drying and other post-treatment purification, 7.3g of beta-NMN is obtained, and the purity is more than 99.9%.
Example 6
Sequentially adding 30L of pure water into a 50L glass reaction kettle, and respectively dissolving 150g/L NR, ATP with a final concentration of 5mM and MgC1 with a final concentration of 10mM 2 Finally, NRK mutant enzyme solution (protein content 5 g/L) prepared by the triple mutant (N28M, A29R, E107A) in the example 1 is added and mixed uniformly, the reaction temperature is set to 30 ℃, the stirring speed is 300rpm, and after the reaction is carried out for 10 hours, the NR conversion rate is 99.93% by HPLC detection. And (3) performing post-treatment purification such as ion exchange resin separation, freeze-drying and the like to obtain 7.28kg of beta-NMN with purity of more than 99.9%.
Example 7
400L of water is added into a 500L reaction kettle, and the temperature is raised to160g/L NR, ATP at a final concentration of 10mM, mgC1 at a final concentration of 15mM were dissolved at 30℃respectively 2 Finally, NRK mutant enzyme solution (protein content 10 g/L) prepared by adding the triple mutant (N28M, A29R, E107A) in the example 1 is uniformly mixed, the stirring speed is set at 200rpm, and after the reaction is carried out for 14 hours, the NR conversion rate is 99.91% by HPLC detection. After ion exchange resin separation, freeze drying and other post-treatment purification, 79.3kg of beta-NMN is obtained, and the purity is more than 99.9%.
From examples 5 to 7, the following conclusions can be drawn: the NRK mutant can convert NR into NMN well under various reaction systems, and compared with wild NRK, the NRK mutant has higher enzyme activity and better stability, and the key is that the conversion rate from NR to NMN is higher, so that the subsequent purification process is greatly facilitated, and NMN products with high purity and higher added value are easier to obtain.
Claims (7)
1. A nicotinamide riboside kinase mutant is characterized by at least one mutation in the amino acid sequence of the nicotinamide riboside kinase mutant compared to the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence derived therefrom that has at least 80% sequence identity.
2. The mutant nicotinamide riboside kinase according to claim 1, wherein the amino acid sequence of the mutant nicotinamide riboside kinase is mutated at least one of positions 24-32 and 102-111 compared to the amino acid sequence shown in SEQ ID No. 1 or an amino acid sequence derived therefrom having at least 80% sequence identity.
3. The mutant nicotinamide riboside kinase according to claim 2, wherein the amino acid sequence of the mutant nicotinamide riboside kinase is mutated at any one of position 28, 29, 103, 107, single, double or triple, as compared to an amino acid sequence derived from the amino acid sequence shown in SEQ ID No. 1, which has at least 80% sequence identity.
4. A nicotinamide riboside kinase mutant according to claim 3, wherein the amino acid sequence of the nicotinamide riboside kinase mutant is subjected to a triple-combination mutation at positions 28, 29, 103, wherein the N at position 28 is M, the a at position 29 is R, and the F at position 103 is S, as compared to the amino acid sequence of SEQ ID No. 1 from which the amino acid sequence is derived having at least 80% sequence identity.
5. Use of a nicotinamide riboside kinase mutant according to any one of claims 1 to 4 for the preparation of a β -nicotinamide mononucleotide.
6. The use of a nicotinamide riboside kinase mutant according to claim 5 for the preparation of a β -nicotinamide mononucleotide, wherein nicotinamide riboside is used as a substrate and ATP is used as a phosphate donor, and the β -nicotinamide mononucleotide is produced by a reaction under the catalysis of the nicotinamide riboside kinase mutant.
7. The use of a nicotinamide riboside kinase mutant according to claim 5 for the preparation of a β -nicotinamide mononucleotide, wherein said nicotinamide riboside kinase mutant is used in combination with a polyphosphate kinase.
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