CN114075582A - Preparation method and application of D-ribose-5-phosphate - Google Patents

Abstract

The invention provides a preparation method of D-ribose-5-phosphate, which comprises the step of carrying out phosphorylation reaction on D-ribose and a phosphate donor to prepare the D-ribose-5-phosphate by using ribokinase, wherein the amino acid sequence of the ribokinase is D-ribokinase shown as NCBI accession numbers WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _ 160803741.1. Also disclosed is an enzyme composition, the contents of which are set forth below. The D-ribose kinase has high stability and enzyme activity, and the preparation of the D-ribose 5-phosphate by using the D-ribose kinase has the advantages of simple process operation, high conversion rate and high production efficiency, and is suitable for higher substrate concentration.

Description

Preparation method and application of D-ribose-5-phosphate

Technical Field

The invention relates to the field of biocatalysis, and in particular relates to a preparation method and application of D-ribose-5-phosphate.

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. NAD + is too large in molecular weight to be taken orally into cells, and is mainly dependent on cellular synthesis in vivo, and the amount synthesized 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 content of the beta-NMN is increased, and the metabolism caused by aging is obviously inhibited, so that the beta-NMN becomes a 'non-aging medicine', 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 of the routes for preparing NMN by a biological enzyme method is to obtain NMN by taking D-ribose and nicotinamide as starting raw materials and carrying out three-step catalytic reaction under the action of ribokinase, phosphoribosyl pyrophosphate synthetase, nicotinamide phosphoribosyl transferase and the like. For example, CN110373397A discloses that NMN is produced under the catalysis of commercially available ribokinase, phosphoribosyl pyrophosphate synthetase, and nicotinamide ribokinase mutant (R189H/S232T/R302K). CN111051520A discloses that NMN is produced under the catalysis of ScRbk, Bsprs, HdNampt and DrPpk, and the reaction is carried out for 48 hours to generate 3.6mM NMN, wherein ScRbk is Rbk (Ribokinase) derived from Saccharomyces cerevisiae (NCBI accession number P25332), but the yield of NMN still needs to be improved.

The important first step in the three-step reaction is that D-ribose is reacted with ribokinase to obtain D-ribose-5-phosphate, so that it is necessary to develop a high-activity ribokinase capable of catalyzing D-ribose to D-ribose-5-phosphate with high efficiency.

Disclosure of Invention

The invention provides a preparation method and application of D-ribose-5-phosphate, aiming at solving the technical problem that the existing production process of beta-nicotinamide mononucleotide cannot realize the defect of low production efficiency because the concentration of raw materials is improved while the high conversion rate is kept. The inventor of the invention unexpectedly discovers four ribokinases with strong catalytic ability by screening, which can catalyze a substrate D-ribose and a phosphate donor such as adenosine triphosphate disodium salt to prepare D-ribose-5-phosphate.

In order to solve the above technical problems, one of the technical solutions of the present invention is: a process for producing D-ribose 5-phosphate, which comprises subjecting D-ribose and a phosphate donor to phosphorylation reaction using ribokinase to produce the D-ribose 5-phosphate, characterized in that the amino acid sequence of the ribokinase is D-ribokinase represented by NCBI accession Nos. WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _ 160803741.1.

Preferably, the nucleotide sequence for coding the ribokinase is shown as SEQ ID NO 1-4.

In a preferred embodiment, the concentration of the D-ribose is between 10g/L and 100g/L, such as 40 g/L.

Preferably, the molar ratio of the phosphate donor to the D-ribose is 1:1000 to 5:1, such as 1.5: 1; and/or the mass ratio of the ribokinase to the D-ribose is 1: 200-1: 10. The phosphate donor is preferably ATP or ATP sodium salt.

More preferably, the pH of the phosphorylation reaction is 6.0 to 8.0, such as 7.0 to 7.5; and/or the temperature of the phosphorylation reaction is 25-40 ℃, for example 35 ℃.

In a preferred embodiment, the preparation method further comprises a step of regeneration of the phosphate donor: phosphorylating a product of the phosphate donor deprived of the phosphate group into a phosphate donor using a phosphotransferase and a phosphate raw material;

preferably, the molar ratio of the phosphoric acid raw material to the D-ribose is 1: 1-2: 1; and/or the product of the phosphate donor is ADP or ADP sodium salt.

In a preferred embodiment, the phosphate donor is ATP, and/or the phosphate source is a phosphate compound, such as disodium acetyl phosphate, dipotassium acetyl phosphate and/or diammonium acetyl phosphate;

preferably, the molar ratio of the phosphate compound to the D-ribose is 1:1, and/or the mass ratio of the phosphotransferase to the phosphate compound is 1:500 to 1:50, for example 1: 163; and/or the reaction temperature for regenerating the phosphoric acid donor is 20-40 ℃, for example 35 ℃; and/or the reaction pH value for regenerating the phosphoric acid donor is 6-8, such as 7.4.

In a preferred embodiment, the phosphotransferase is acetate kinase having NCBI accession number AAC 75356.1.

Preferably, the nucleotide sequence encoding the acetate kinase is shown as SEQ ID NO 9.

In a preferred embodiment, the preparation of the ribokinase comprises the steps of:

(1) culturing the engineering bacteria containing the ribokinase gene until OD600 is 0.5-1.0, preferably 0.8, inducing at 25 ℃ preferably with IPTG with final concentration of 0.1mM, and culturing for 12-24 hours, preferably 16 hours;

(2) collecting the thalli, resuspending the thalli into a buffer solution according to the weight volume ratio of 1 (5-20), such as 1:10, and crushing to obtain a crude enzyme solution; preferably the crude enzyme solution is purified, for example using Ni BestaroseHP resin, and/or the buffer is 50mM Tris-HCl pH7.5 or PBS buffer; wherein the weight to volume ratio is g: mL.

In order to solve the above technical problems, the second technical solution of the present invention is: provided is a use of ribokinase in the preparation of D-ribose 5-phosphate or β -nicotinamide mononucleotide, wherein said ribokinase is a D-ribokinase having NCBI accession numbers WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _ 160803741.1.

Preferably, the nucleotide sequence for coding the ribokinase is shown as SEQ ID NO 1-4.

In order to solve the technical problems, the third technical scheme of the invention is as follows: providing an enzyme composition comprising:

1) at least two of the D-ribokinases having amino acid sequences represented by NCBI accession numbers WP _054396858.1, CCX07771.1, WP _125988536.1 and WP _ 160803741.1; or,

2) d-ribokinase and phosphotransferase; wherein the D-ribokinase is a D-ribokinase with an amino acid sequence shown in NCBI accession No. WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _160803741.1, and the phosphotransferase is an acetate kinase with an amino acid sequence shown in NCBI accession No. AAC 75356.1.

Preferably, the nucleotide sequence for coding the nicotinamide riboside kinase is shown as SEQ ID NO 1-4; and/or the nucleotide sequence for coding the acetate kinase is shown as SEQ ID NO. 9.

The positive progress effects of the invention are as follows:

the D-ribose kinase has high stability and enzyme activity, and the preparation of the D-ribose-5-phosphate by using the D-ribose kinase has the advantages of simple process operation, high conversion rate and high production efficiency, and is suitable for higher substrate concentration.

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 china biotechnology limited liability, beijing dingding. D-ribose (purchased from Shanghai Michelin Biotechnology, Inc.); ATP was purchased from Cuiguer biotech Inc., Anhui.

EXAMPLE 1 preparation of D-Ribokinase (Ribokinase, Rbk)

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 and KH₂PO₄2.31g/L、K₂HPO₄·2H₂Dissolving O16.43 g/L in deionized water, diluting to desired volume, and sterilizing at 121 deg.C for 20 min.

The ribokinase amino acid information is shown in Table 1 below, and is synthesized from the whole gene of Min-No. 698, Songjiang province, Shanghai, Biotechnology engineering (Shanghai), the enzyme cutting sites NdeI and HindIII, and the vector pET28 a. The synthesized D-ribokinase gene is transformed into host E.coli BL21(DE3) competent cells to obtain an engineering strain containing the ribokinase gene. Wherein Enz.01 is SEQ ID NO. 7 of CN 111051520A; enz.02 is Bioorganic & Medicinal Chemistry 14(2006), 6327-6332 medium ribokinase, NCBI accession number AAC 76775.1.

TABLE 1D-Ribose kinase

Engineering bacteria containing Rbk 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 the inoculation amount of 1% (v/v), shake-cultured at 37 ℃ until the OD600 reaches about 0.8, cooled to 25 ℃, added with IPTG to the final concentration of 0.1mM, and induced-cultured for 16 h. After the culture, the culture solution was collected and centrifuged at 4000rpm at 4 ℃ for 20min (Centrifuge: Eppendorf Centrifuge 5810R), the supernatant was discarded, and the cells were collected and stored in an ultra-low temperature refrigerator at-20 ℃ for further use.

3g of the collected thallus is taken out and resuspended in 30mL of 50mM Tris-HCl buffer solution with pH 7.4, the high-pressure homogenization and crushing are carried out at 4 ℃ to obtain a ribokinase crude enzyme solution, the homogenized ribokinase crude enzyme solution is purified by Ni BestaroseHP (Boglong biotech Co., Ltd., product number AA0041) resin, the protein concentration is measured by a Bradford kit (purchased from GmbH, Czeri bioengineering Co., Ltd., Shanghai) and the ribokinase crude enzyme solution is stored in a refrigerator at-20 ℃ for standby. Protein concentrations are shown in table 2.

TABLE 2

The results of sequence homology alignment of ribokinase with prior art enz.01 and enz.02, respectively, are shown in table 3 below:

TABLE 3

The enzyme sequences have low sequence homology with ribokinases that have been disclosed in the prior art for the preparation of NMN.

EXAMPLE 2 preparation of acetate kinase

The acetate kinase cleavage sites NdeI, HindIII, vector pET28a were synthesized from the acetate kinase (NCBI accession No. AAC75356.1) gene (SEQ ID NO: 9). 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 was completed, the culture was centrifuged at 4500rpm for 20min (Centrifuge: Eppendorf Centrifuge 5810R), the supernatant was discarded, and the cells were collected and stored in an ultra-low temperature refrigerator at-20 ℃ for further use.

Taking 10g of acetate kinase to be resuspended in 50mL of 0.1M sodium phosphate buffer solution with pH7.5, 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 protein concentration was 15 mg/mL.

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

adenosine Diphosphate (ADP) 4.0g, diammonium acetyl phosphate 1.38g, anhydrous magnesium chloride 0.19g, 10mL of 0.2M sodium phosphate buffer pH7.5 and 70mL of pure water were added, dissolved by magnetic stirring, adjusted to pH7.5 with 20% Na2CO3 aqueous solution, and the volume was adjusted to 100mL of the substrate solution. 19.5mL of the substrate solution was placed in a reaction vessel and incubated for 10min at 150rpm in a shaker at 30 ℃. 0.5ml of diluted acetate kinase liquid enzyme was added thereto, and the mixture was reacted at 30 ℃ in a shaker at 150 rpm. After reacting for 10min, taking reaction liquid, adding 2M hydrochloric acid, shaking up to quench reaction, detecting generated Adenosine Triphosphate (ATP) by an ion-pair HPLC method, calculating the ATP concentration according to a standard curve, and calculating the enzyme activity of liquid enzyme to be 10048U/mL.

Ion pair HPLC detection method:

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 3 preparation of D-ribose 5-phosphate

48mL of pure water was added to the reaction system, 2g D-ribose (13.3mmol), 0.20g of magnesium chloride hexahydrate, and 11g of adenosine disodium triphosphate (19.9mmol) were added thereto, the mixture was dissolved in a water bath at 25 ℃ under stirring, the pH was adjusted to about 7.4 with a 4% aqueous solution of sodium hydroxide (in terms of mass/volume), and finally 2mL of a pure enzyme solution of ribokinase was added thereto. 2H was reacted at 35 ℃ and pH7.0-7.5 at 150rpm, and the progress of the reaction was checked by TLC during the reaction, and the results are shown in Table 4.

And (3) a TLC detection method:

developing agent: ethyl acetate, isopropanol, water, acetic acid 3:3:1: 1; color developing agent: 10% sulfuric acid ethanol (5.5mL +90mL ethanol mix); the gun was baked at 250 ℃ for 15 seconds.

D ribose Rf ═ 0.7; R5P Rf is 0.2. The results show that the Enz.04, Enz.05, Enz.07 and Enz.08 can all obtain better conversion rate than the Enz.01 in the prior art, wherein the conversion rate of the Enz.04, Enz.07 and Enz.08 can almost reach 100 percent.

TABLE 4 preparation of ribose-5-phosphate by liquid enzyme catalysis

Enzyme	Conversion rate
		Enz.01	<50％
Enz.04	100％
		Enz.05	About 50 percent
Enz.07	100％
		Enz.08	100％

Example 4 Effect of different pH on the reaction

48mL of pure water was added to the reaction system, 2g D-ribose (13.3mmol), 0.20g of magnesium chloride hexahydrate, and 11g of adenosine disodium triphosphate (19.9mmol) were added thereto, the mixture was dissolved in a water bath at 25 ℃ with stirring, the pH was adjusted to different values with 4% aqueous sodium hydroxide (in terms of mass/volume), and finally 2mL of pure enzyme solutions were added. 2H was reacted at 35 ℃ and 150rpm, and the progress of the reaction was checked by TLC during the reaction, and the results are shown in Table 5.

TABLE 5

pH	Enz.04	Enz.05	Enz.07	Enz.08
					6.0	100％	About 50 percent	100％	100％
7.0	100％	About 50 percent	100％	100％
					8.0	100％	About 50 percent	100％	100％

The results show that the reaction proceeds well under the conditions of pH6.0 to 8.0.

Example 5 Effect of different temperatures on the reaction

48mL of pure water was added to the reaction system, 2g D-ribose (13.3mmol), 0.20g of magnesium chloride hexahydrate, and 11g of adenosine disodium triphosphate (19.9mmol) were added thereto, the mixture was dissolved in a water bath at 25 ℃ with stirring, the pH was adjusted to 7.0 with a 4% aqueous solution of sodium hydroxide (in terms of mass/volume), and finally 2mL of pure enzyme solutions were added. The reaction was carried out at 150rpm for 2H at different temperatures, and the progress of the reaction was checked by TLC during the reaction, and the results are shown in Table 6.

TABLE 6

Temperature of	Enz.04	Enz.05	Enz.07	Enz.08
					25℃	100％	About 50 percent	100％	100％
30℃	100％	About 50 percent	100％	100％
					35℃	100％	About 50 percent	100％	100％
40℃	100％	About 50 percent	100％	100％

The result shows that the reaction can be well carried out under the temperature of 25-40 ℃.

EXAMPLE 6 preparation of D-ribose 5-phosphate

48mL of pure water was added to the reaction system, 2g D-ribose (13.3mmol), 0.20g of magnesium chloride hexahydrate, 0.2g of adenosine disodium triphosphate, and 2.45g of disodium acetyl phosphate (13.3mmol) were added thereto, the mixture was dissolved in a water bath at 25 ℃ under stirring, the pH was adjusted to about 7.4 with a 4% aqueous solution of sodium hydroxide (mass/volume ratio), and finally 2mL of a pure ribokinase enzyme solution and 1mL of a pure acetokinase enzyme solution were added thereto, respectively. 2H was reacted at 35 ℃ and pH7.0 to 7.5 at 150rpm, and the progress of the reaction was checked by TLC and TCL was checked as in example 3, and the results are shown in Table 7.

TABLE 7

Enzyme	Conversion rate
		Enz.01	<50％
Enz.04	100％
		Enz.05	About 50 percent
Enz.07	100％
		Enz.08	100％

The difference between this example and example 3 is that the latter provides excess ATP disodium salt (19.9mmol), whereas this example regenerates the phosphate donor with acetate kinase, with the ATP disodium salt content being only 2mmol, but with a catalytic effect comparable to example 3, indicating that acetate kinase can regenerate the phosphate donor (ATP) reaction product very well.

SEQUENCE LISTING

<110> Chongkola Biotechnology (Shanghai) Ltd

<120> preparation method and application of D-ribose-5-phosphate

<130> P20014545C

<160> 9

<170> PatentIn version 3.5

<210> 1

<211> 918

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.04

<400> 1

atgaacaccg ttaccgttat cggttctatc aacctggacc gtaccatccg tgttgaaaac 60

atgccgaaac cgggtgaaac catccacacc aaagaaatct tctctgctgg tggtggtaaa 120

ggtgctaacc aggctgttgc tgctcagcgt tctggtgcta aaacccactt catcggtgct 180

gttggtgacg acgctgctgg taaaaccatg ctggacctgc tgacccagga aaaaatcaac 240

ctggctggta tcaccaaaat gaccaaccag tctaccggtc aggcttacgt taccgttgac 300

gacgctggtg aaaaccagat catgatccac ggtggtgcta acatggcttt caccccggct 360

gacgttgaag ctcaccgtga catcatcgaa gcttctgact tcgttgttgc tcagttcgaa 420

tctgctgttg actctaccgt tgaagctttc aaaatcgctc aggctgctgg tgttcgtacc 480

atcctgaacc cggctccggc tatggaaaaa gttccggctg aactgctggc tgttaccgac 540

atgatcgttc cgaacgaaac cgaaaccgaa accctgaccg gtatcgctat caccgacgaa 600

gcttctatgc tgaaagcttc tgctgctctg cacgctctgg gtatctctgc tgttatcatc 660

accatcggtt ctaaaggtgc tttctacgac atcgacggtc gtcacggtat cgttccggct 720

ttcaaagttg aagctgttga caccacctct gctggtgaca ccttcatcgg tgctatgtct 780

tctgttctga acaaagactt ctctaacctg gaagacgcta tccgttacgg taaccgtgct 840

tcttctatcg ctgttcagcg tttcggtgct cagccgtcta tcccgtacaa aaacgaaatc 900

accgctgctg aaggtaaa 918

<210> 2

<211> 918

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.05

<400> 2

atgtctatcc tgatcatcgg ttctctgaac accgacctga tcaccgttac cccgcgtatg 60

ccggctgctg gtgaaaccct gaccgcttct tctttccaca ccgctccggg tggtaaaggt 120

gctaaccagg ctgttgcttg cgctcgtctg tctctgccgc cgaccaaagt ttctatgatc 180

ggttctgttg gtcgtgactc tttcgcttct ccgctgctgt ctgaactgga agactctggt 240

gttgacgttg ctcgtgttac ctctcacacc tctgaaccga ccggttctgc tgttatcgtt 300

gttgacggtt ctaacggtga aaaccgtatc ctgatccaca ccggtgctaa cggtaccgtt 360

accccggaat gcttcccgga aaccgaagac tggaacgctg ttgttatgca gctggaaatc 420

ccggttccga ccgttctgtc tatcctgtct gctgctaaaa cccagggtgt taaaaccatc 480

ttcaacccgg ctccggctgt tccgctgccg gaagaatgct ggaaagacgt tgactggtgc 540

atcgttaacg aaaccgaagc tgctatcctg accgctgttg aacagaacgt tctggactct 600

aaagaaggtg ttgaaaccgc tggtaaagaa ctgctgcgtc gtggttgcgg tgctgttgtt 660

gttaccctgg gtgctcgtgg tgcttactgg tgcgctgaag acgaagaagg ttgggttgaa 720

accggtgtta aaaaagaaga cgttgttgac tctaccggtg ctggtgacac cttcgttggt 780

gctctggctg ttggtgttgt tgaaggtaaa aaacgtgaag aatgcgttga cttcgctcgt 840

cgtgctgctg gtcgtgctgt tcgtaaacgt ggtgctatgg ctggtgttcc gtggcgtcgt 900

gaagttgaag aaggtgtt 918

<210> 3

<211> 903

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.07

<400> 3

atggaaaaca tcctggttgt tggttctatg aacatggacc tggttgttaa caccgaccgt 60

gttccggaca aaggtgaaac catcatcggt aaatctttcg aacaggttcc gggtggtaaa 120

ggtgctaacc aggctgctgc tgttggtaaa ctgggtggtc gtgtttcttt cgtttctgct 180

tgcggtaaag actctttcgg tgacgacctg ctgtcttctc tgcaggacaa aggtgttgac 240

acctcttctg ttttcaccct ggacgacaac accggtatcg ctgctatcac cgttgaagaa 300

gacggtgaca accgtatcat cgttgttcag ggtgctaacg cttctctgtc tccggaaatg 360

atcgaccagg ttgaaggtaa aatcaaagaa gctgcttacc tgctgctgca gatggaaatc 420

ccgctggcta ccgttatcca caccatcgaa ctggctgact tctaccagac ccgtgttatc 480

ctggacccgg ctccggctca gggtctgccg cgtgaaatct actctaaaat cgactacctg 540

ctgccgaact ctggtgaact ggctctgctg ctggaagaat acgacctgcg tgacgaagaa 600

gacaaaatcg gtcagctgct ggactggggt gttaaaaaca tcctgatcac caaaggttct 660

gaaggtgtta ccctgtacca gaaaggttct cagcaggttt acccgaccct gaaagttaaa 720

gctgttgaca ccaccgctgc tggtgacacc ttcgctggtg ctctggcttt cggtctgcag 780

aaaggttggg acatcgaccg ttgcatctct ttcggtaacc gtgctgctgc tatctctgtt 840

acccgtgctg gtgctcagtc ttctatcccg tctttcgctg aagttgaaaa catgaaaggt 900

atc 903

<210> 4

<211> 915

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.08

<400> 4

atgaaaaaag ttgttgttct gggttctctg aacatggacc tgtctatcga aaccaaccgt 60

atgccgcgta acggtgaaac catcgacggt aaatctttct tcatgtctcc gggtggtaaa 120

ggtgctaacc aggctgttgc tgctcagaaa tctggtgctc cgacctctat gatcggttct 180

gttggtaacg acctgttcgg ttctcagctg atctcttctc tgaaaaaaga aggtgttgac 240

tgcacccacg ttatggaaaa cgactctacc tctaccggta tcgctatgat catccgtaac 300

gctggtgaca accgtatcat cctgggttct ggtgctaact acaccgttga cgaagcttac 360

acctctcagg ctctgcagga aatcgctaac aaagaagaca tcttcctgac ccagttcgaa 420

tctgactacg aagttgttct gcactctctg gctaaagcta aatctcaggg tctgttcacc 480

gttttcaacc cggctccggc taaagacatc ccggaaaccg cttacccgtc tatcgacctg 540

ctgatcgtta accagtggga atctgaaatg ctgtctggta tctacccgaa aaccgacaaa 600

gactgcgaag acgctatcaa actgttcctg gacaaaggtg tttcttctgt tatcatcacc 660

tgcggtgctg ctggttctac ctacggtgac aaagaacagc tgatcttcgt tccgtctttc 720

aaaaccaacg ttgttgacac caccgctgct ggtgacacct acatcggtgc tctggttgct 780

tctctggcta acgaaaccac catgaaagac tctatgatct acgctaccaa agctgcttct 840

ctggctatct ctaaacaggg tgctcaggaa tctatcccgt acaaaaacga aatcgaccag 900

ttcaaagaag ttaac 915

<210> 5

<211> 999

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.01

<400> 5

atgggtatca ccgttatcgg ttctctgaac tacgacctgg acaccttcac cgaccgtctg 60

ccgaacgctg gtgaaacctt ccgtgctaac cacttcgaaa cccacgctgg tggtaaaggt 120

ctgaaccagg ctgctgctat cggtaaactg aaaaacccgt cttctcgtta ctctgttcgt 180

atgatcggta acgttggtaa cgacaccttc ggtaaacagc tgaaagacac cctgtctgac 240

tgcggtgttg acatcaccca cgttggtacc tacgaaggta tcaacaccgg taccgctacc 300

atcctgatcg aagaaaaagc tggtggtcag aaccgtatcc tgatcgttga aggtgctaac 360

tctaaaacca tctacgaccc gaaacagctg tgcgaaatct tcccggaagg taaagaagaa 420

gaagaatacg ttgttttcca gcacgaaatc ccggacccgc tgtctatcat caaatggatc 480

cacgctaacc gtccgaactt ccagatcgtt tacaacccgt ctccgttcaa agctatgccg 540

aaaaaagact gggaactggt tgacctgctg gttgttaacg aaatcgaagg tctgcagatc 600

gttgaatctg ttttcgacaa cgaactggtt gaagaaatcc gtgaaaaaat caaagacgac 660

ttcctgggtg aataccgtaa aatctgcgaa ctgctgtacg aaaaactgat gaaccgtaaa 720

aaacgtggta tcgttgttat gaccctgggt tctcgtggtg ttctgttctg ctctcacgaa 780

tctccggaag ttcagttcct gccggctatc cagaacgttt ctgttgttga caccaccggt 840

gctggtgaca ccttcctggg tggtctggtt acccagctgt accagggtga aaccctgtct 900

accgctatca aattctctac cctggcttct tctctgacca tccagcgtaa aggtgctgct 960

gaatctatgc cgctgtacaa agacgttcag aaagacgct 999

<210> 6

<211> 927

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.02

<400> 6

atgcagaacg ctggttctct ggttgttctg ggttctatca acgctgacca catcctgaac 60

ctgcagtctt tcccgacccc gggtgaaacc gttaccggta accactacca ggttgctttc 120

ggtggtaaag gtgctaacca ggctgttgct gctggtcgtt ctggtgctaa catcgctttc 180

atcgcttgca ccggtgacga ctctatcggt gaatctgttc gtcagcagct ggctaccgac 240

aacatcgaca tcaccccggt ttctgttatc aaaggtgaat ctaccggtgt tgctctgatc 300

ttcgttaacg gtgaaggtga aaacgttatc ggtatccacg ctggtgctaa cgctgctctg 360

tctccggctc tggttgaagc tcagcgtgaa cgtatcgcta acgcttctgc tctgctgatg 420

cagctggaat ctccgctgga atctgttatg gctgctgcta aaatcgctca ccagaacaaa 480

accatcgttg ctctgaaccc ggctccggct cgtgaactgc cggacgaact gctggctctg 540

gttgacatca tcaccccgaa cgaaaccgaa gctgaaaaac tgaccggtat ccgtgttgaa 600

aacgacgaag acgctgctaa agctgctcag gttctgcacg aaaaaggtat ccgtaccgtt 660

ctgatcaccc tgggttctcg tggtgtttgg gcttctgtta acggtgaagg tcagcgtgtt 720

ccgggtttcc gtgttcaggc tgttgacacc atcgctgctg gtgacacctt caacggtgct 780

ctgatcaccg ctctgctgga agaaaaaccg ctgccggaag ctatccgttt cgctcacgct 840

gctgctgcta tcgctgttac ccgtaaaggt gctcagccgt ctgttccgtg gcgtgaagaa 900

atcgacgctt tcctggaccg tcagcgt 927

<210> 7

<211> 918

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.03

<400> 7

atgaacaaag ttaccgttct gggttctctg aacgttgaca ccatcctgcg tttcaaacgt 60

ttcccgaaac cgggtgaaac cctgccgctg accggtaaat ctgttgctgg tggtggtaaa 120

ggtgctaacc aggctatcgc tgctgctcgt gctggtgctc agaccacctt catcggtaaa 180

gttggtaccg accaggaagg taccttcatg gttcagcagc tgaccgactc tggtgttgac 240

gaccagtacg ttcagcactc tgacgctgct aacaccggtt ctgctttcat cctgctggac 300

tcttcttctg aaaaccgtat cctgatcgac ggtggtacca accagcaggt taccgctaaa 360

gacgttgaac gtgctcagtc tgctatctct acctctacct tcctgatcgc tcagttcgaa 420

accccgatcg ctgctaccat ccgtggtttc gaactggctc aggctgctgg tcagaaaacc 480

atcctgaacc cggctccggc taccaccacc gttccggctg aactgctggc taccaccgac 540

ctgatcgttc cgaacgaaac cgaaaccgaa accctgaccg gtgttcacgt taccgacgaa 600

ccgtctatga tccagggtgc tcagaaactg caggctctgg gtgttgctaa cgttatcatc 660

accgttggtt ctaaaggtgc tttctggatg cgtggtgctg aacacggttt cgttgctgct 720

tacaaagttg aagctgttga caccaccgct gctggtgaca ccttcatcgg tgctctgtct 780

tctgttctga tgccggactt ctctaacctg gctgctgctg ttcgtttcgc taaccgtgct 840

tcttctatcg ctgttcagaa actgggtgct cagccgtcta tcccgaccaa agaagctatc 900

gaagctgctg aacgtgct 918

<210> 8

<211> 876

<212> DNA

<213> Artificial Sequence

<220>

<223> Enz.06

<400> 8

atgcgtcagc cgcgtatcac cgttgttggt tctatcaaca tggacctggt tacctctacc 60

gacatcttcc cgcagcaggg tgaaaccgtt cgtggtgaag acttccacac caacccgggt 120

ggtaaaggtg ctaaccaggc tgttgctgct gctcgtctgg gtgctgacgt tcacatggtt 180

ggtcgtgttg gtgacgacac cttcggtgaa aacctgctgc acaacctgca gcaggaaaac 240

atcgacacct ctatggttga ccaggttgct aacacctctt ctggtctggc taacatcacc 300

ctgtctgaaa aagacaaccg tatcatcatc atcccgggtg ctaacaacca ggttaccccg 360

gaatacgtta aagcttgcga agaaaccatc ctggcttctg actacgttct gctgcagttc 420

gaaatcccga aagaaaccat cgaatactgc atcgacgttt gcgctcagca cgacatcccg 480

gttgttgtta acccggctcc ggttatgccg ctgtctccgg accagtggga aaaagcttct 540

gttatcaccc cgaacgaaac cgaagctgct gaactgttca aaggtcgtta cgacgaaatc 600

caggaaaaac tggttatcac caaaggtaaa gaaggtgttg aattcttcga ccacggtacc 660

cagaaaaacg tttctccgta caaagttgaa gttgctgaca ccaccggtgc tggtgacacc 720

ttcaacggtg ctctggctgt tgctctggct gaaggtcaga ccctgaccgc tgctgttcag 780

ttcgctaacg ctgctggtgc tctgtctgtt cagaaactgg gtgctcaggg tggtatgccg 840

acccgtcagg ctgttgacca gatgctggaa gaacgt 876

<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 (10)

1. A process for producing D-ribose 5-phosphate, which comprises subjecting D-ribose and a phosphate donor to phosphorylation reaction using ribokinase to produce the D-ribose 5-phosphate, wherein the ribokinase has an amino acid sequence represented by NCBI accession Nos. WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _ 160803741.1; preferably, the nucleotide sequence for coding the ribokinase is shown as SEQ ID NO 1-4.

2. The process according to claim 1, wherein the concentration of D-ribose is 10g/L to 100g/L, such as 40 g/L.

3. The method according to claim 1, wherein the molar ratio of the phosphate donor to the D-ribose is 1:1000 to 5:1, such as 1.5: 1; and/or the mass ratio of the ribokinase to the D-ribose is 1: 200-1: 10; preferably, the phosphate donor is ATP or ATP sodium salt.

4. The method of claim 1, wherein the phosphorylation reaction has a pH of 6.0 to 8.0, such as 7.0 to 7.5; and/or the temperature of the phosphorylation reaction is 25-40 ℃, for example 35 ℃.

5. The method of any one of claims 1-4, further comprising the step of performing a phosphate donor regeneration: phosphorylating a product of the phosphate donor deprived of the phosphate group into a phosphate donor using a phosphotransferase and a phosphate raw material;

preferably, the molar ratio of the phosphoric acid raw material to the D-ribose is 1: 1-2: 1; and/or the product of the phosphate donor is ADP or ADP sodium salt.

6. The method according to claim 5, wherein the phosphate donor is ATP, and/or the phosphate source is a phosphate compound such as disodium acetyl phosphate, dipotassium acetyl phosphate and/or diammonium acetyl phosphate;

preferably, the molar ratio of the phosphate compound to the D-ribose is 1:1, and/or the mass ratio of the phosphotransferase to the phosphate compound is 1:500 to 1:50, for example 1: 163; and/or the reaction temperature for regenerating the phosphoric acid donor is 20-40 ℃, for example 35 ℃; and/or the reaction pH value for regenerating the phosphoric acid donor is 6-8, such as 7.4.

7. The method according to claim 5 or 6, wherein the phosphotransferase is acetate kinase having NCBI accession No. AAC 75356.1; preferably, the nucleotide sequence encoding the acetate kinase is shown as SEQ ID NO 9.

8. The process according to any one of claims 1 to 7, wherein the ribokinase is prepared by the steps of:

(1) culturing the engineering bacteria containing the ribokinase gene until OD600 is 0.5-1.0, preferably 0.8, inducing at 25 ℃ preferably with IPTG with final concentration of 0.1mM, and culturing for 12-24 hours, preferably 16 hours;

(2) collecting the thalli, resuspending the thalli into a buffer solution according to the weight volume ratio of 1 (5-20), such as 1:10, and crushing to obtain a crude enzyme solution; preferably, the crude enzyme solution is purified, for example using NiBestaroseHP resin, and/or the buffer is 50mM Tris-HCl pH7.5 or PBS buffer; wherein the weight to volume ratio is g: mL.

9. Use of a ribokinase in the preparation of D-ribose 5-phosphate or β -nicotinamide mononucleotide, wherein said ribokinase is a D-ribokinase with NCBI accession numbers WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _ 160803741.1; preferably, the nucleotide sequence for coding the ribokinase is shown as SEQ ID NO 1-4.

10. An enzyme composition, comprising:

1) at least two of the D-ribokinases having amino acid sequences represented by NCBI accession numbers WP _054396858.1, CCX07771.1, WP _125988536.1 and WP _ 160803741.1; or,

2) d-ribokinase and phosphotransferase; wherein the D-ribokinase is a D-ribokinase with an amino acid sequence shown in NCBI accession No. WP _054396858.1, CCX07771.1, WP _125988536.1 and/or WP _160803741.1, and the phosphotransferase is an acetate kinase with an amino acid sequence shown in NCBI accession No. AAC 75356.1;

preferably, the nucleotide sequence for coding the nicotinamide riboside kinase is shown as SEQ ID NO 1-4; and/or the nucleotide sequence for coding the acetate kinase is shown as SEQ ID NO. 9.