CN114075585A

CN114075585A - Preparation method of beta-nicotinamide mononucleotide

Info

Publication number: CN114075585A
Application number: CN202010832445.1A
Authority: CN
Inventors: 程占冰; 王舒; 田振华; 焦琦
Original assignee: Ecolab Biotechnology Shanghai Co ltd
Current assignee: Ecolab Biotechnology Shanghai Co ltd
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22

Abstract

The invention discloses a preparation method of beta-nicotinamide mononucleotide. The preparation method utilizes nicotinamide riboside kinase with an amino acid sequence shown as NCBI accession numbers XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1 and/or PMB71451.1 to carry out phosphorylation reaction on nicotinamide ribose and/or nicotinamide riboside chloride in a system with a phosphate donor to obtain the beta-nicotinamide mononucleotide. The preparation method of beta-nicotinamide mononucleotide of the invention realizes the effects of improving the concentration of the substrate, maintaining high conversion rate and greatly shortening the catalytic time, is favorable for large-scale production of beta-nicotinamide mononucleotide by using nicotinamide ribose as a raw material and reduces the production cost.

Description

Preparation method of beta-nicotinamide mononucleotide

Technical Field

The invention relates to the field of biocatalysis, and in particular relates to a preparation method of beta-nicotinamide mononucleotide.

Background

beta-Nicotinamide mononucleotide (beta-Nicotinamide monouclotide, beta-NMN or NMN) is NAD⁺(nicotinamide adenine dinucleotide, also known as coenzyme 1) the most direct precursor, NAD⁺Is an important active substance participating in thousands of important physiological reactions such as cell metabolism, oxidation reduction, protein transcription and the like. Due to NAD⁺The molecular weight is too large to be taken into cells by oral administration, and its in vivo synthesis is mainly dependent on cellular synthesis and the amount of synthesis is very low. But with the addition of NAD⁺Research on precursor small molecular substance beta-NMN shows that eating beta-NMN can effectively promote in vivo NAD⁺The beta-NMN can obviously inhibit the aging of the organism, so that the beta-NMN becomes a medicine for treating the uneasiness, and has great development potential and market prospect in the aspect of functional health-care food. At present, NMN is approved as a raw material of health food in developed countries such as Europe, America, Japan, and the like, and a plurality of health care products such as American HeRBALmax, GeneHarbor NMN9000, Japan MIRAI LAB NMN3000 capsule, Australian synext, and the like are developed by taking NMN as a main component.

One route for preparing NMN by a biological enzyme method is to obtain NMN by taking D-ribose and nicotinamide as initial raw materials and carrying out three-step catalytic reaction under the action of ribokinase, phosphoribosyl pyrophosphate synthetase, nicotinamide phosphoribosyl transferase and the like. The route has more intermediate products and difficult subsequent separation and purification, so the overall yield is low and the production cost is high.

Another route for preparing NMN by a biological enzyme method is a method for preparing beta-nicotinamide mononucleotide by taking D-5-phosphoribosyl and nicotinamide as raw materials and under the catalysis of Phosphoribosyl Pyrophosphate Synthetase (PRPPs) and nicotinamide phosphoribosyl transferase (NAMPT) in the presence of ATP, for example, CN2018109407295 discloses the method, and the conversion rate of a substrate can reach 86.9% -89.8%. For another example, WO2018/023206 discloses that NMN is prepared from nicotinamide and 5 '-phosphoribosyl-1' -pyrophosphate (PRPP) as raw materials under the catalytic action of a nicotinamide phosphoribosyltransferase mutant (F180A), and the conversion rate of nicotinamide as a substrate can reach 66%.

The other route is that Nicotinamide Ribose (NR) is used as a starting material, and under the action of Nicotinamide riboside kinase (NrK) and ATP, NMN is obtained through one-step reaction, and the yield and the product purity are high. Among them, CN2016112456194 uses Nicotinamide Ribose (NR) as a substrate, and reacts for 24 hours in the presence of phosphate donors such as ATP and under catalysis of nicotinamide ribokinase derived from Saccharomyces cerevisiae (Saccharomyces cerevisiae) to generate β -nicotinamide mononucleotide, and high performance liquid chromatography detects that nicotinamide ribose conversion rate is above 90%, purity of β -nicotinamide mononucleotide after purification is greater than 95%, but concentration of nicotinamide as the substrate is only 18 mM. Patent CN2019107231777 discloses a nicotinamide riboside kinase NrK derived from Kluyveromyces marxianus and a mutant (D45E/D58Q/R161K/Y164W) gene thereof, and provides a construction method of an enzyme-exogenous expression system and a construction method of an enzyme mutant, and a method for preparing nicotinamide mononucleotide by catalyzing NR by using the enzyme and the mutant as a biocatalyst. After 20h of reaction, the conversion rate of NR (NR) of the substrate is more than 80 percent by HPLC (high performance liquid chromatography), the purity of the beta-nicotinamide mononucleotide obtained after purification is more than 98 percent, the concentration of the nicotinamide as the substrate is up to 50g/L, but the conversion rate is reduced from 90 percent to 80 percent along with the increase of the substrate concentration from 10g/L to 50 g/L.

However, in the prior art, the conversion rate and the substrate feeding concentration of the methods are still to be improved; and the reaction time is too long, which is not beneficial to large-scale production and application.

Disclosure of Invention

The technical problems to be solved by the invention are that aiming at the problems that in the existing method for producing beta-nicotinamide mononucleotide by taking nicotinamide riboside as starting material, the concentration of nicotinamide riboside as a substrate can not be improved, and simultaneously, higher substrate conversion rate can not be kept, and the required reaction time is too long, which is not beneficial to improving the production efficiency of beta-nicotinamide mononucleotide and being applied to large-scale production, the inventor has unexpectedly found through a plurality of experiments that a plurality of nicotinamide riboside kinases can keep higher substrate conversion rate while improving the concentration of nicotinamide riboside as a substrate, and the required catalytic time is greatly shortened; therefore, a method for producing beta-nicotinamide mononucleotide is provided, which can increase the concentration of substrate and maintain higher substrate conversion rate, and the required catalytic time is greatly shortened.

In order to solve the above problems, one of the technical solutions provided by the present invention is: a process for the preparation of β -nicotinamide mononucleotide, comprising phosphorylating nicotinamide riboside and/or nicotinamide riboside chloride with nicotinamide riboside kinase and a phosphate donor to obtain said β -nicotinamide mononucleotide, said nicotinamide riboside kinase being nicotinamide riboside kinase having an amino acid sequence as represented by NCBI accession Nos. XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1 and/or PMB 71451.1. Preferably, the nucleotide sequence encoding the nicotinamide riboside kinase is the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and/or SEQ ID NO. 8.

Preferably, the concentration of the nicotinamide riboside and/or nicotinamide riboside chloride is 5 g/L-150 g/L; preferably, the concentration of nicotinamide riboside chloride is 100 g/L.

Preferably, the phosphate donor is ATP or ATP sodium salt; the mass ratio of the phosphate donor to the nicotinamide riboside and/or nicotinamide riboside chloride is 1: 1000-3: 1. In order to fully convert nicotinamide ribochloride, in a preferred embodiment of the invention, the mass ratio of the phosphate donor ATP sodium salt to nicotinamide ribochloride is 5: 2.

The inventors found that when the substrate is excessive, the reaction efficiency is lowered; whereas the use of an excess of nicotinamide riboside kinase results in a waste of enzyme, so:

preferably, the mass ratio of the nicotinamide riboside kinase to the nicotinamide riboside and/or nicotinamide riboside chloride is 1: 2000-1: 60. Preferably, the mass ratio of said nicotinamide riboside kinase to said nicotinamide riboside chloride is 1: 350.

Preferably, the phosphorylation reaction temperature is 20-40 ℃, and preferably 25 ℃. The pH value of the phosphorylation reaction is 6-8, and preferably 7.0.

In order to save cost and further improve reaction efficiency due to the higher cost of phosphate donors such as ATP, the preparation method preferably further comprises the step of regenerating the phosphate donor: the phosphate donor product from which the phosphate group has been lost is phosphorylated to a phosphate donor by using a phosphotransferase and a phosphate starting material. In this case, the mass ratio of the phosphate donor such as ATP to nicotinamide ribochloride is preferably 1: 10.

Preferably, the phosphotransferase is an acetate kinase; when the phosphate donor is ATP or ATP sodium salt, the phosphate donor product that has lost phosphate groups is ADP or ADP sodium salt.

According to the present invention, the phosphoric acid raw material is preferably a phosphoric acid compound. The phosphate compound may be selected from acetyl phosphate including disodium acetyl phosphate, dipotassium acetyl phosphate and/or diammonium acetyl phosphate.

According to the invention, in order to ensure a sufficient reaction, the phosphate donor is preferably present in excess of the substrate; the molar ratio of said phosphoric acid starting material to said nicotinamide riboside and/or nicotinamide riboside chloride is at least 1: 1.

Preferably, the preparation of said nicotinamide riboside kinase or said phosphotransferase comprises the steps of:

(1) culturing the engineering bacteria containing the nicotinamide riboside kinase or the phosphotransferase to OD600 of 0.5-1.0, inducing at 25 ℃, preferably with IPTG with final concentration of 0.1mM, and culturing for 12-24 hours; in a preferred embodiment of the invention, the engineering bacteria is E.coli BL21(DE3), the engineering bacteria are cultured until OD600 is 0.8, and the culture time is 16 hours;

(2) collecting thalli, resuspending the thalli into a buffer solution according to a weight-volume ratio of 1: 5-1: 10, and crushing to obtain a crude enzyme solution, wherein the unit of the weight-volume ratio is g/ml; preferably, the crude enzyme solution is purified, for example with a Ni BestaroseHP resin, and/or the buffer is a 50mM sodium phosphate or Tris-HCl buffer, pH 7.5.

Preferably, the amino acid sequence of the acetate kinase is as shown in NCBI accession number AAC 75356.1; more preferably, the nucleotide sequence for coding the acetate kinase is the nucleotide sequence shown in SEQ ID NO. 9.

Preferably, the mass ratio of the acetate kinase to the acetyl phosphate is 1: 300-1: 20; in a preferred embodiment of the present invention, when the phosphate raw material is acetyl phosphate disodium salt, the mass ratio of the acetate kinase to the acetyl phosphate disodium salt is 1: 86.

Preferably, the reaction temperature of the step of regenerating the phosphoric acid donor is 20-40 ℃, and the preferable temperature is 25 ℃;

and/or the reaction pH value of the step of regenerating the phosphoric acid donor is 6-8, and the preferable reaction pH value is 7.0.

In order to solve the above problems, the second technical scheme provided by the invention is as follows: the nicotinamide riboside kinase of the invention is used as a catalyst in the preparation of beta-nicotinamide mononucleotide.

In order to solve the above problems, the third technical solution provided by the present invention is: an enzyme composition, comprising:

1) at least two of the aforementioned nicotinamide riboside kinases of the invention;

or 2) the enzyme composition comprises the above-described nicotinamide riboside kinase of the invention and the above-described phosphotransferase of the invention.

Unless otherwise specified, concentrations and ratios of the reactants described herein refer to the initial concentrations and initial ratios of the materials in the overall system of the reaction.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: when the concentration of the substrate nicotinamide riboside reaches 100g/L, the substrate conversion rate is not lower than 95%, and the reaction time is greatly shortened from more than 20h in the prior art to not more than 6 h; after the step of regeneration of the phosphate donor is added, the conversion rate of the substrate is still not lower than 95% after 6h of catalysis under the condition that the concentration of the substrate nicotinamide riboside is 100g/L, at the moment, the dosage of the phosphate donor is reduced, and the production cost is greatly reduced.

Drawings

FIG. 1 is a graph of the catalytic preparation of NMN using nicotinamide riboside kinase having NCBI accession number XP-023459718.1;

FIG. 2 is a diagram of a reaction initial reaction solution;

FIG. 3 is a substrate NRC control map;

figure 4 is a product NMN control profile;

FIG. 5 is a nicotinamide control profile;

FIG. 6 is an ADP control profile;

FIG. 7 is a map of AMP control.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

The experimental methods in the invention are conventional methods unless otherwise specified, and the gene cloning operation can be specifically referred to the molecular cloning experimental guidance compiled by J. Sambruka et al.

pET28a and a protein extraction reagent (bugbuster protein extraction reagent) were purchased from Novagen; the DpnI enzyme Purchase from England Weiji (Shanghai) trade, Inc.; NdeI enzyme, HindIII enzyme were purchased from Thermo Fisher, e.coli bl21(DE3) competent cells were purchased from changsheng biotechnology, llc, beijing dingding. Nicotinamide Riboside Chloride (NRC) (purchased from McRoche Biotech, Suzhou).

The ion pair HPLC detection method involved in the specific embodiment is as follows:

a chromatographic column: welch Ultimate AQ-C18(5 μm, 4.6 × 250 mm); buffer solution: 0.05mol/L diammonium hydrogen phosphate aqueous solution: 10% tetrabutylammonium hydroxide aqueous solution 91: 1 (volume ratio), and adjusting the pH to 3.6 by phosphoric acid; mobile phase: acetonitrile: buffer 8: 92 (volume ratio); flow rate: 1.0 mL/min; detection wavelength: 254 nm; column temperature: 30 ℃; sample introduction amount: 10 mu l of the mixture; operating time: 40 min; diluting liquid: ultrapure water.

EXAMPLE 1 preparation of NR kinase

Composition of LB liquid medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, dissolving with deionized water, fixing the volume, and sterilizing at 121 ℃ for 20min for later use.

TB liquid medium composition: 10g/L of peptone, 18g/L of yeast powder, 4mL/L of glycerol, KH2PO42.31g/L, K2HPO 4.2H 2O 16.43.43 g/L, dissolving with deionized water, fixing the volume, and sterilizing at 121 ℃ for 20min for later use.

The amino acid and nucleotide sequence information of NR kinase is shown in Table 1 below. The NR kinase (Enz.01-Enz.20) gene is synthesized by Suzhou Jinwei Zhi Biotech limited (Ministry of New area, Jiangjing, Jiangbei, Yongshu, Ministry of Japan, Yangguan No. 211) through the whole gene, the enzyme cutting sites NdeI and HindIII and the vector pET28 a. The synthesized NR kinase gene was transformed into host e.coli BL21(DE3) competent cells to obtain an engineered strain containing the NR kinase gene.

TABLE 1 NR kinase information Table

Engineering bacteria containing NR kinase genes are respectively streaked and activated on a plate, and then a single colony is selected and inoculated into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and shake culture is carried out for 4h at 37 ℃. Transferred to 150mL of fresh TB liquid medium containing 50. mu.g/mL of kanamycin at an inoculation amount of 1 v/v%, shake-cultured at 37 ℃ until OD600 reaches about 0.8, and IPTG was added to a final concentration of 0.1mM, and induced-cultured at 25 ℃ for 16 hours. After the culture is finished, the culture solution is centrifuged at 4500rpm for 20min, supernatant is discarded, and thalli are collected and stored in an ultra-low temperature refrigerator at minus 20 ℃ for standby.

Taking 10g of the collected thallus, suspending the collected thallus in 50mL of 0.1M sodium phosphate buffer solution with pH7.5, homogenizing and crushing under high pressure to obtain an NR kinase crude enzyme solution, purifying the homogenized crude enzyme solution by using Ni BestaroseHP (Boglong (Shanghai) Biotechnology Co., Ltd., product number AA0041) resin, measuring the protein concentration by using a Bradford kit (purchased from Shanghai Czeri bioengineering Co., Ltd.), and storing in a refrigerator at-20 ℃ for later use.

The enzyme activity determination method comprises the following steps:

to 1mL of the reaction system were added 29.2mg of substrate NRC (100mM), 57mg of ATP disodium salt (100mM), 1.9mg of MgCl₂·6H₂O (20mM), 20. mu.L of NR kinase enzyme solution, 980. mu.L of 20mM phosphate buffer pH7.5, followed by reaction at 30 ℃ in a shaker at 200rpm, and the specific parameters of the reaction system are shown in Table 2 below.

TABLE 2 reaction conditions

After reaction is carried out for 4 times by continuously sampling 100 mu L every 15min, diluting the solution by 10 times with dilute hydrochloric acid with pH of about 2.0, detecting by ion pair HPLC, calculating the concentration of NMN according to a standard curve of the product NMN, and calculating the enzyme activity, wherein the results are shown in the following table 3.

The enzyme activity is defined as: the amount of enzyme required to produce 1. mu. moL NMN per minute at 30 ℃ at pH7.5 is 1 enzyme activity unit (1U).

TABLE 3 liquid enzyme Activity

EXAMPLE 2 preparation of NMN by NR kinase

57mL of pure water was added to the reaction system, 0.30g of magnesium chloride hexahydrate, 15g of adenosine disodium triphosphate were added, the mixture was dissolved in a 25 ℃ water bath with stirring, the pH was adjusted to about 8.2 with a 4% sodium hydroxide aqueous solution, 6.0g of Nicotinamide Ribochloride (NRC) as a substrate was added, the pH was adjusted to 7.2 to 7.5 with a 20% sodium carbonate aqueous solution after dissolution, and 3mL of the liquid enzyme of example 1 was added. The reaction was carried out for 6H at 150rpm in a 25 ℃ water bath at pH6.5-7.0, and the substrate conversion was determined by ion-pair HPLC during the reaction, the results of which are shown in Table 4.

Conversion calculation formula:

conversion rate-amount of NRC converted/amount of NRC starting × 100%

The detection pattern of the Enz.06 conversion rate is shown in figure 1. The reaction initial reaction liquid map is shown in figure 2, the substrate NRC control map is shown in figure 3, the product NMN control map is shown in figure 4, the nicotinamide control map is shown in figure 5, the ADP control map is shown in figure 6, and the AMP control map is shown in figure 7.

TABLE 4 liquid enzyme catalyzed preparation of NMN

The results show that the catalytic efficiency of Enz.02, Enz.03, Enz.06, Enz.07, Enz.10, Enz.14, Enz.16 and Enz.18 is high, and the conversion rate can reach more than 95% after 6 hours of reaction.

EXAMPLE 3 preparation of acetate kinase

The acetate kinase gene, the restriction sites NdeI and HindIII, and the vector pET28a were synthesized from the acetate kinase (NCBI accession No. AAC75356.1, nucleotide sequence shown in SEQ ID NO:9) gene. The synthesized acetate kinase gene is transformed into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing the acetate kinase gene.

After the engineering bacteria containing the acetate kinase gene are respectively streaked and activated on a plate, a single colony is selected and inoculated into 5mL LB liquid culture medium containing 50 mug/mL kanamycin, and shake culture is carried out for 4h at 37 ℃. Transferred to 150mL of fresh TB liquid medium containing 50. mu.g/mL of kanamycin at an inoculation amount of 1 v/v%, shake-cultured at 37 ℃ until OD600 reaches about 0.8, and IPTG was added to a final concentration of 0.1mM, and induced-cultured at 25 ℃ for 16 hours. After the culture is finished, the culture solution is centrifuged at 4500rpm for 20min, supernatant is discarded, and thalli are collected and stored in an ultra-low temperature refrigerator at minus 20 ℃ for standby.

Taking 10g of acetate kinase to be resuspended in 50ml of 0.1M sodium phosphate buffer solution with pH value of 7.5, homogenizing and crushing at high pressure to obtain crude enzyme solution of the acetate kinase, purifying the homogenized crude enzyme solution by using Ni BestaroseHP (Boglong (Shanghai) Biotechnology Co., Ltd., product number AA0041) resin, measuring the protein concentration by using a Bradford kit (purchased from Shanghai Czeri bioengineering Co., Ltd.), and storing in a refrigerator at-20 ℃ for later use.

The method for measuring the enzyme activity of the acetate kinase liquid enzyme comprises the following steps:

in a reaction vessel, 4.0g of Adenosine Diphosphate (ADP), 1.38g of acetyl diammonium phosphate, 0.19g of anhydrous magnesium chloride, 10mL of 0.2M sodium phosphate buffer solution having a pH of 7.5 and 70mL of pure water were added, dissolved by magnetic stirring, and dissolved with 20% (g/mL) of Na₂CO₃The pH of the aqueous solution was adjusted to 7.5 and the volume was adjusted to 100mL of substrate solution. Taking 19.5mL of substrate solution to be placed in a reaction container, placing the reaction container in a 30 ℃ shaking table at 150rpm for 10min, adding 0.5mL of diluted acetate kinase, and reacting in the shaking table at 30 ℃ and 150 rpm. After the reaction is carried out for 10min, 2M hydrochloric acid is added into reaction liquid, shaking is carried out to quench the reaction, generated Adenosine Triphosphate (ATP) is detected by an ion-pair HPLC method, the ATP concentration is calculated according to a standard curve, and the enzyme activity of liquid enzyme is calculated to be 10048U/mL, and the protein concentration is 15 mg/mL.

EXAMPLE 4 preparation of NMN by regeneration of ATP

54mL of pure water, 0.30g of magnesium chloride hexahydrate, 0.6g of adenosine disodium triphosphate (ATP disodium salt) and 3.86g of disodium acetyl phosphate were added to the reaction system, the mixture was dissolved in a water bath at 25 ℃ with stirring, the pH was adjusted to about 8.2 with 4% (g/mL) of an aqueous solution of sodium hydroxide, 6.0g of a substrate, Nicotinamide Ribochloride (NRC), was added thereto, the pH was adjusted to 7.2 to 7.5 with 20% (g/mL) of an aqueous solution of sodium carbonate, and 3mL of an acetic acid kinase and 3mL of a kinase liquid enzyme were added, respectively. Reacting in a water bath at 25 ℃ and pH6.5-7.0 for 6h at 150rpm, and measuring the substrate conversion rate by ion pair HPLC in the reaction process, wherein the results are shown in Table 5.

TABLE 5

As can be seen from the above experiments, the substrate nicotinamide ribochloride with a concentration of 100g/L can be converted by more than 95% within not more than 6h by using the method of the present invention.

After the ATP regeneration step is added, the conversion of the substrate nicotinamide riboside chloride with the concentration of 100g/L within no more than 6h can be achieved, but the use amount of ATP is greatly reduced from 15g to 0.6g, so that the production cost is greatly reduced.

SEQUENCE LISTING

<110> Chongkola Biotechnology (Shanghai) Ltd

<120> preparation method of beta-nicotinamide mononucleotide

<130> P20014327C

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.02

<400> 1

atgccgccga aaaccgttat cgttggtgtt tctggtgctt cttgctctgg taaatctacc 60

ctgacccagt tcctgcacgc tatcttcaaa ggttcttcta tcctgcacga agacgacttc 120

tacaaaaccg acgctgacat cccggttaaa aacggtatcg ctgactggga ctgcaaagaa 180

tctctgaacc tggacctgtt cctggaaacc ctgaaataca tccgtgaaca cggtgacctg 240

ccgacccacc tgcacaaccg tgacaacaaa aacgttgcta aagaagctct ggctcagttc 300

acctctatcc tggaagaata cccggaatct ccgatctctt cttctgacta caaaatcatc 360

ttcgttgacg gtttcatgct gtacgttaac gaagaactga tccgttcttt cgacctgtgc 420

ctgatgctgg tttgcgactt cgacaccctg cgtgctcgtc gtgaatctcg taaaggttac 480

gttacccagg aaggtttctg gcaggacccg ccgaactact tcgaaaacta cgtttggccg 540

ggttacgtta acggtcacgc tcacctgttc cagaacaaag acgttcacgg taaactggtt 600

gacacccgta tccagctgtc tccgtcttct aaactgtctg ttaaacagaa cgttgaatgg 660

gctgttgcta ccatccagaa agctctgtct 690

<210> 2

<211> 690

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.03

<400> 2

atgccgccga aaaccgttat cgttggtgtt tctggtgctt cttgctctgg taaatctacc 60

ctgacccagt tcctgcacgc tatcttcaaa ggttcttcta tcctgcacga agacgacttc 120

tacaaaaccg acgctgacat cccggttaaa gacggtgttc cggactggga ctgcaaagaa 180

tctctgaacc tggactcttt cctggaaacc ctgaaataca tccgtaaaca cggtgacctg 240

ccgacccacc tgcacaaccg tgacaacctg aacgttgcta aagaagctct gacccagtac 300

aacgaaatcc tgaaagaata cccggaatct ccgatctctt cttctgacta caaattcatc 360

ttcgttgacg gtttcatgct gtacgttaac caggaactga tccagtcttt cgacctgtgc 420

ctgatgctgg cttgcgactt cgacaccctg aaagctcgtc gtgaatctcg taaaggttac 480

gttacccagg aaggtttctg gcaggacccg ccgaactact tcgaaaacaa cgtttggccg 540

ggttacgtta acggtcactc tcacctgttc gaaaacaaag acgtttacgg taaactgatc 600

gactcttcta tccagctgtc tccggtttct aacatgtctg ttcagcagaa cgttcagtgg 660

gctatcaaca ccatccagaa attcgtttct 690

<210> 3

<211> 762

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.06

<400> 3

atgccgatca cccgtggtga caaaaaagct atcctggttg gtatctctgg tgtttcttct 60

tctggtaaaa ccaccctggc tcgtatcctg cgtgacatct tcccgcgtgc tttcatcctg 120

cacgaagacg acttctacaa aaccgacgaa cagatcccga tccacccgga acacaaaatc 180

gctgactggg actgcctgga atctatcaac ctgccggctt tcgaaaaagc tctgaaacac 240

atccgtgaca acggtctgtc tccgccggac ttcacctcta aagaagacac caactctgtt 300

ggtgaagttg aagttgacaa agctctgatc gaagacctga aatggaaagc ttctaaatgg 360

atgttcgctg acgctccgtc tatcgctatc atcgacggtt tcctgctgta ctctgaaggt 420

atgaaacagg ttcgtgacct gttcgacgtt aaactgttcc tgcgtaccga ctaccagacc 480

gctaaagctc gtcgtgaagc tcgtaccggt tacgttacca tcgaaggttt ctgggaagac 540

ccgccgggtt acgttgacaa aatcgtttgg ccgaactacg ttaaagacca cgctttcatg 600

ttcaaagacg gtaacgttga aggtaccctg gacgaccgtg ttctgaaaga cctggacatc 660

aaaaccatgc cggacgaagc tccggctgac atgacctctt tcgttaaatg ggcttacgac 720

gttttcgaag aagctctgca ggacttcacc atgccgcgtg ac 762

<210> 4

<211> 760

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.07

<400> 4

catatgtctg cgaacaaagc gctgatcgtg ggcatcagcg gctgcagcag ctctggcaaa 60

accaccctgg cgcgtctgct gcgtgatatt ttcccgaaca ccttcatcct gcatgaagat 120

gacttttacc gcccggaaaa tgaactgccg accaaaaacg gcctgctgga ctgggactgt 180

gcggaagcgc tggacatccc ggcgatggcg gaatccctgg cgtacatccg tcagcacgca 240

gcgttcccgc cgactctgga tagcaaagaa gatcagaact ccgtgggcaa atgcccggtt 300

tctgaagcga gcattgcagc gcagcgtgcg aaagttgacg cggcactggg tccggaccac 360

ccgctgcgta aaaacctgcg cctgtgcctg ctggatggtt tcctgctgta tagcccgtcc 420

atggcagcga ttaaaccgaa cctggatatc aaactgttcc tgcgtaccac ctacgaaaaa 480

gcgaaaaaac gtcgtgaagc tcgcgatggt tacgtaaccc tggaaggctt ctgggctgat 540

ccgccgggct acgttgataa aatcgtttgg ccgaactatg ttgaagaaca tgcgtggatg 600

ttcgaatacg gtgacgttga aggtaaattt aaaactgatg ttctggataa agaaggtatt 660

aaagttcagt ctgacgtgag cgcggatggt gatatcgaaa aaaccttcga atggaccgtt 720

gataccgttc tggaagaact gggtaaacag gtgtaagctt 760

<210> 5

<211> 750

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.10

<400> 5

atgggtacca acaaagctct gatcgctggt atctctggtt gctcttcttc tggtaaaacc 60

accctggctc gtctgctgcg tgacatcttc ccgcacacct tcatcctgca cgaagacgac 120

ttctaccgtc cggaaaccga actgccgacc aaaaacggtc tgctggactg ggactgcgct 180

gaagctatcg acatcccggc tatggctgaa tctctggctt acatccgtca gcacgctgct 240

ttcccgccga ccctggactc taaagaagac cagaacttcg ttggtaaatg cccggtttct 300

gaagctacca tcgctgctca gcgtgctaaa gttgacgctg ctctgggtcc ggaccacccg 360

ctgcgtaaca acctgcgtct gtgcctgttc gacggtttcc tgctgttctc tccgtctatg 420

gctgctatca aaccgtctct ggacatcaaa ctgttcctgc gtaccaccta cgaaaaagct 480

aaaaaacgtc gtgaagctcg tgacggttac gttaccctgg aaggtttctg ggctgacccg 540

ccgggttacg ttgacaaaat cgtttggccg aactacgttg aagaacacgc ttggatgttc 600

gaaggtggtg acgttgaagg taaattcaaa accgacgttc tggacaacga aggtatcaaa 660

atccagtctg acgttgacgc tgacggtgac atcgaaaaaa ccttcgaatg gaccgttgac 720

accatcctga aagaactgaa agaccaggtt 750

<210> 6

<211> 753

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.14

<400> 6

atggctgaca aaaaagctct gatcgttgct ctgtctggtt gctcttcttc tggtaaaacc 60

accctggctc gtctgctgcg tgacatcttc ccgtctacct tcatcctgca cgaagacgac 120

ttctaccgtc cggaaaccga actgccgaaa aaagacgacc tgctggactg ggactgcgct 180

gaagctatcg acatcccggc tatggctgaa accctgtctt acatccgtca gcacgctgct 240

ttcccgccga ccctggactc ttaccaggac aaaaactctg ttggtgaatg cccggttccg 300

cagtctacca tctctgctct gaaatctaaa gtttcttctc tgctgccgtc tacccacccg 360

ctggctacct ctgctctgca cctgtgcatc ctggacggtt tcctgctgta cccgccgtct 420

atgtctgcta tccagccgca cctggacatc aaaatcttcc tgcgtgcttc ttacgctcag 480

gctaaagctc gtcgtgaagc tcgtgacggt tacgttaccc tggaaggttt ctgggctgac 540

ccgccgggtt acgttgacaa aatcgtttgg ccgaactacg ttgctgaaca ctcttggatg 600

ttccaggacg gtgacgttga aggtgaatac aaagctgacg ttctggaacg tgaagctatc 660

cgtgttccgg gtggttctgg tgttgacggt gaaatggaca aaatcctgga atggatggtt 720

gacctgatcc tggaagaaat gaaaaaacac gct 753

<210> 7

<211> 807

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.16

<400> 7

atggctcagg acgacaaacg tgctctgatc ctgggtatct ctggttgctc ttcttctggt 60

aaaaccaccc tggctcgtct gctgcgtgac atcttcccgt ctaccttcat cctgcaccag 120

gacgacttct accgtccgga aaccgaactg ccgtctaaaa acggtctgct ggactgggac 180

tgcgctgaag ctatcgacgt tgttgctatg gctgacgctc tggcttacat ccgtcagcac 240

gctgaattcc cgccgaccct ggactctaaa gaagaccaga actctgttgg taaatgcccg 300

gttccggact ctaccatcgc tgctctgaaa gctaaagttt cttctgctct gccgccgtct 360

cacccgctgc tgccgcgtgg tccgtcttct ggttctaact ctggttctgg tgctgacccg 420

gttccgtctc tgcgtctgtg cctgctggac ggtttcctgc tgtacggtcc gtctatggct 480

gctctgcgtt cttctttcga cgttaaactg ttcctgcgtg cttcttacgc tcgtgctaaa 540

gctcgtcgtg aagctcgtga cggttacgtt accctggaag gtttctgggc tgacccgccg 600

ggttacgttg acgctatcgt ttggccgaac tacgttgaag aacacgcttg gatgttcgaa 660

tctggtgacg ttgaaggtcg tttccgtgac gaagttctgc gtgctgaagg tatccgtgtt 720

ctggaaggtg ctccggttga cgctgacatg gaacagctgc tggaatggat ggttgacctg 780

atcctggaag aaatgcgtaa actgcag 807

<210> 8

<211> 765

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.18

<400> 8

atgggtgacc gtaaagctct ggttgttgct atctctggtt gctcttcttc tggtaaaacc 60

accctgtctc gtctgctgcg tgacatcttc ccgtctacct tcatcctgca cgaagacgac 120

ttctacaaaa ccgacaaaga catcccgatc aacaacggtg ttgctgactg ggactgcgct 180

gaagctatcg acatcccggc tatgaccgaa gctgttgctt acatccgtca gcacgctgaa 240

atcccgccga ccctgaactc tatcgaagac cagaactctg ttggtaaaaa cccggttccg 300

gacgctctga tcaacgaact gaaagctcgt gtttctgctg ctatcccgtc ttctcacccg 360

ctgcgtgacg acgacgacgc tcgtctgctg cgtctgtgcc tgttcgacgg tttcctgctg 420

tactctaaat ctatgaccga actggctccg tctctggaca tcaaaatcct gctgcgtgct 480

tctcacgctc aggctaaagc tcgtcgtgaa gctcgtgacg gttacgttac cctggaaggt 540

ttctggaaag acccgccggg ttacgttgac cacgttgttt ggccgaacta cgttaaagaa 600

cacgcttggc tgttcgaaaa cggtgacgtt gaaggtgaat accgtcagga agttctgcag 660

accgaaggta tcgttgttcc gaaaggtggt gctctggacc cggacatggc tgaaaccctg 720

cagtggatgg ttgacatcat cctgaccgaa ctgcagcgtc acaac 765

<210> 9

<211> 1200

<212> DNA

<213> Artificial Sequence

<220>

<223> acetate kinase

<400> 9

atgtcttcta aactggttct ggttctgaac tgcggttctt cttctctgaa attcgctatc 60

atcgacgctg ttaacggtga agaatacctg tctggtctgg ctgaatgctt ccacctgccg 120

gaagctcgta tcaaatggaa aatggacggt aacaaacagg aagctgctct gggtgctggt 180

gctgctcact ctgaagctct gaacttcatc gttaacacca tcctggctca gaaaccggaa 240

ctgtctgctc agctgaccgc tatcggtcac cgtatcgttc acggtggtga aaaatacacc 300

tcttctgttg ttatcgacga atctgttatc cagggtatca aagacgctgc ttctttcgct 360

ccgctgcaca acccggctca cctgatcggt atcgaagaag ctctgaaatc tttcccgcag 420

ctgaaagaca aaaacgttgc tgttttcgac accgctttcc accagaccat gccggaagaa 480

tcttacctgt acgctctgcc gtacaacctg tacaaagaac acggtatccg tcgttacggt 540

gctcacggta cctctcactt ctacgttacc caggaagctg ctaaaatgct gaacaaaccg 600

gttgaagaac tgaacatcat cacctgccac ctgggtaacg gtggttctgt ttctgctatc 660

cgtaacggta aatgcgttga cacctctatg ggtctgaccc cgctggaagg tctggttatg 720

ggtacccgtt ctggtgacat cgacccggct atcatcttcc acctgcacga caccctgggt 780

atgtctgttg acgctatcaa caaactgctg accaaagaat ctggtctgct gggtctgacc 840

gaagttacct ctgactgccg ttacgttgaa gacaactacg ctaccaaaga agacgctaaa 900

cgtgctatgg acgtttactg ccaccgtctg gctaaataca tcggtgctta caccgctctg 960

atggacggtc gtctggacgc tgttgttttc accggtggta tcggtgaaaa cgctgctatg 1020

gttcgtgaac tgtctctggg taaactgggt gttctgggtt tcgaagttga ccacgaacgt 1080

aacctggctg ctcgtttcgg taaatctggt ttcatcaaca aagaaggtac ccgtccggct 1140

gttgttatcc cgaccaacga agaactggtt atcgctcagg acgcttctcg tctgaccgct 1200

Claims

1. A method for the preparation of β -nicotinamide mononucleotide, comprising the phosphorylation reaction of nicotinamide riboside and/or nicotinamide riboside chloride using nicotinamide riboside kinase and a phosphate donor to obtain said β -nicotinamide mononucleotide, characterized in that said nicotinamide riboside kinase is nicotinamide riboside kinase having an amino acid sequence as represented by NCBI accession No. XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1 and/or PMB 71451.1; preferably, the nucleotide sequence encoding the nicotinamide riboside kinase is the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and/or SEQ ID NO. 8.

2. The method according to claim 1, wherein the concentration of nicotinamide riboside and/or nicotinamide riboside chloride is 5g/L to 150 g/L; preferably, the concentration of nicotinamide riboside chloride is 100 g/L.

3. The method of claim 1, wherein the phosphate donor is ATP or ATP sodium salt; the mass ratio of the phosphate donor to the nicotinamide riboside and/or nicotinamide riboside chloride is 1: 1000-3: 1; preferably, the mass ratio of the ATP sodium salt to nicotinamide ribochloride is 5: 2.

4. The method according to claim 1, wherein the mass ratio of nicotinamide riboside kinase to nicotinamide riboside and/or nicotinamide riboside chloride is 1:2000 to 1: 60; preferably, the mass ratio of said nicotinamide riboside kinase to said nicotinamide riboside chloride is 1: 350.

5. The method according to claim 1, wherein the phosphorylation reaction temperature is 20 to 40 ℃; preferably, the temperature of the phosphorylation reaction is 25 ℃;

and/or the pH value of the phosphorylation reaction is 6-8; preferably, the pH of the phosphorylation reaction is 7.0.

6. The method of any one of claims 1-5, further comprising the step of regenerating the phosphate donor: the phosphate donor product without phosphate group is phosphorylated into phosphate donor by using phosphotransferase and phosphate raw material; when the step of regenerating the phosphate donor is included, the mass ratio of the phosphate donor to nicotinamide riboside chloride is preferably 1: 10;

preferably, the phosphotransferase is acetate kinase, and the phosphate donor product that loses its phosphate group is ADP or an ADP sodium salt; and/or the phosphoric acid raw material is a phosphoric acid compound; more preferably, the phosphate compound is an acetyl phosphate, including disodium acetyl phosphate, dipotassium acetyl phosphate, and/or diammonium acetyl phosphate;

even more preferably, the preparation of said nicotinamide riboside kinase or said phosphotransferase comprises the steps of:

(1) culturing an engineered bacterium containing said nicotinamide riboside kinase or said phosphotransferase to an OD600 of 0.5-1.0, preferably 0.8, said engineered bacterium preferably being E.coli BL21(DE3), inducing at 25 ℃, preferably with IPTG at a final concentration of 0.1mM, and culturing for 12-24 hours, preferably 16 hours;

7. The method according to claim 6, wherein the amino acid sequence of the acetate kinase is represented by NCBI accession No. AAC 75356.1;

preferably, the mass ratio of the acetate kinase to the acetyl phosphate is 1: 300-1: 20;

more preferably, the acetyl phosphate is disodium acetyl phosphate, and the mass ratio of the acetate kinase to the disodium acetyl phosphate is 1: 86; and/or the nucleotide sequence for coding the acetate kinase is the nucleotide sequence shown as SEQ ID NO. 9.

8. The method according to claim 6 or 7, wherein the reaction temperature in the step of regenerating the phosphate donor is 20 to 40 ℃; preferably, the reaction temperature of the step of regenerating the phosphate donor is 25 ℃;

and/or the reaction pH value of the regeneration step of the phosphoric acid donor is 6-8; preferably, the reaction pH of the step of regenerating the phosphate donor is 7.0.

9. Use of a nicotinamide riboside kinase having an amino acid sequence as represented by NCBI accession nos. XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1 and/or PMB71451.1 as a catalyst in the preparation of beta-nicotinamide mononucleotide; preferably, the nucleotide sequence encoding the nicotinamide riboside kinase is the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and/or SEQ ID NO. 8.

10. An enzyme composition, comprising:

1) at least two of the nicotinamide riboside kinases having amino acid sequences as set forth in NCBI accession nos. XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1, and PMB 71451.1;

or 2) the enzyme composition comprises nicotinamide riboside kinase and phosphotransferase; wherein said nicotinamide riboside kinase is a nicotinamide riboside kinase having an amino acid sequence as set forth in NCBI accession No. XP _013025941.1, XP _013017940.1, XP _023459718.1, TVY65083.1, KAF4999805.1, XP _024756901.1, XP _018180784.1 and/or PMB71451.1, and said phosphotransferase is an acetate kinase having an amino acid sequence as set forth in NCBI accession No. AAC 75356.1;

preferably, the nucleotide sequence encoding the nicotinamide riboside kinase is the nucleotide sequence shown as SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and/or SEQ ID NO. 8; and/or the nucleotide sequence for coding the acetate kinase is the nucleotide sequence shown as SEQ ID NO. 9.