A nicotinamide phosphoribosyltransferase mutant and its application in preparing β-nicotinamide mononucleotide

Abstract

The present invention provides a nicotinamide phosphoribosyltransferase (Nampt) mutant and its application in the preparation of β-nicotinamide mononucleotide (β-Nicotinamide Mononucleotide, referred to as NMN), belonging to the field of enzyme engineering technology. The present invention screens nicotinamide phosphoribosyltransferases from different sources and finds that the mutant from Diplococcus cellulosus ( Deinococcus cellulosilyticus ) has the highest Nampt activity. After mutation, it was found that the Nampt[Dc] E227N D334M double mutant had the highest activity, which was 1.9 times that of the wild type. The nicotinamide phosphoribosyltransferase and its mutants were used as biocatalysts for the enzymatic synthesis of NMN. The catalyst conversion efficiency can reach up to 71%, and the NMN yield can reach up to 29.24g / L.

Description

Technical Field

[0001] The present invention belongs to the technical field of enzyme engineering, and specifically relates to a nicotinamide phosphoribosyltransferase mutant and an application thereof in the preparation of β-nicotinamide mononucleotide. Background Art

[0002] β-Nicotinamide mononucleotide (β-NMN) plays an important role in the energy production of human cells and is a + NAD is one of the key precursors of + It can directly or indirectly affect a variety of cellular processes that play a vital role in maintaining homeostasis in the body. As people age, NAD + Levels decline, a decline that is associated with age-related diseases, and restoring NAD + levels, these age-related diseases can be delayed or even reversed.

[0003] But NAD + The molecular weight is too large, and the oral absorption rate is low. + Research on the precursor small molecule β-NMN found that taking β-NMN can effectively increase NAD in the body + The latest research shows that oral NMN promotes NAD + The life span of biological model systems close to humans can be extended by more than 30% [1] This discovery has prompted researchers in the global pharmaceutical, food, and cosmetics industries to continue researching and developing β-NMN.

[0004] One is to use nicotinamide riboside (NR) as the starting material, and in the presence of ATP, nicotinamide ribokinase (NRK) catalyzes the production of NMN. The NMN yields reported so far are low, and the substrate raw materials are expensive; the other is to use nicotinamide and 5-phosphoribosyl-1-pyrophosphate (PRPP) as substrates to prepare NMN under the catalysis of nicotinamide phosphoribosyltransferase (Nampt), among which PRPP is expensive in the market and its source is limited, resulting in a high production cost for this biocatalytic method.

[0005] Patent CN115927513A provides a one-pot method using D-ribose, ATP, nicotinamide, etc. as substrates, ribokinase, phosphoribosyl pyrophosphokinase, nicotinamide phosphoribosyltransferase, and polyphosphate kinase as catalysts, and multiple enzymes for co-catalysis. The method uses lower-cost D-ribose as a raw material to replace the expensive PRPP. At the same time, the one-pot method is simple to operate compared with the traditional step-by-step feeding, and the reactants can continuously undergo multi-step reactions, which shortens the reaction time, improves the reaction efficiency, and reduces the cost. However, the yield is low, only 29.9 mg / L.

[0006] Patent CN117230037A provides a kind of Mannheimia sp. ) The mutant modified nicotinamide phosphoribosyltransferase uses D-ribose, ATP, nicotinamide, etc. as substrates and ribokinase, pyrophosphohydrolase, phosphoribosyl pyrophosphate synthetase, adenylate kinase, polyphosphate kinase, and nicotinamide phosphoribosyltransferase mutant as catalysts. The final molar yield is 92%, and the converted yield is 21.84 g / L.

[0007] At present, in the bioenzymatic synthesis of β-NMN, Nampt is the most critical rate-limiting enzyme, which can catalyze the reaction of nicotinamide and ribose pyrophosphate to synthesize β-NMN. Therefore, improving the catalytic efficiency of nicotinamide phosphoribosyltransferase is a key factor in reducing the biocatalytic synthesis cost of β-NMN, improving the industrial application value of nicotinamide phosphoribosyltransferase, and promoting the application of biocatalytic technology in the industrial production of β-NMN.

[0008] [1]Das A, Huang GX, Bonkowski MS, et al. Impairment of an EndothelialNAD+-H2S Signaling Network Is a Reversible Cause of Vascular Aging [publishedcorrection appears in Cell. 2019 Feb 7;176(4):944-945. Summary of the invention

[0009] In order to solve the above problems, the present invention provides a nicotinamide phosphoribosyltransferase mutant and its application in the preparation of β-nicotinamide mononucleotide.

[0010] In one aspect, the present invention provides a nicotinamide phosphoribosyltransferase mutant.

[0011] Specifically, the nicotinamide phosphoribosyltransferase mutant is obtained by performing point mutations at position 227 and / or position 334 of the nicotinamide phosphoribosyltransferase shown in SEQ ID NO: 1, wherein the nicotinamide phosphoribosyltransferase shown in SEQ ID NO: 1 is derived from Diplococcus cellulosus ( Deinococcus cellulosilyticus NBRC 106333 ), referred to as Nampt[Dc] (GenBank: GEM46430.1).

[0012] More specifically, the mutation at position 227 includes: E227V, E227Y, E227I, E227M, E227K, E227D, E227C, E227T, E227R, E227S, E227G, E227A, E227P, E227W, E227Q, E227F, E227H, E227L or E227N.

[0013] Preferably, the mutation at position 227 includes: E227S, E227N, E227Q or E227A.

[0014] More specifically, the mutation at position 334 includes: D334V, D334Y, D334I, D334M, D334K, D334E, D334C, D334T, D334R, D334S, D334G, D334A, D334P, D334W, D334Q, D334F, D334H, D334L or D334N.

[0015] Preferably, the mutation at position 334 includes: D334Y, D334M, D334S, D334A, D334F or D334N.

[0016] More specifically, the point mutations at positions 227 and 334 include: E227AD334Y, E227SD334Y, E227QD334Y, E227ND334Y, E227AD334M, E227SD334M, E227QD334M, E227ND334M, E227AD334S, E227SD334S, E227QD334Y, E227ND334Y 34S, E227ND334S, E227AD334A, E227SD334A, E227QD334A, E227ND334A, E227AD334F, E22 7SD334F, E227QD334F, E227ND334F, E227AD334N, E227SD334N, E227QD334N and E227ND334N

[0017] Preferably, the point mutations at positions 227 and 334 include: E227A and D334M, E227N and D334M, E227Q and D334S, E227Q and D334A or E227S and D334N.

[0018] On the other hand, the present invention provides a gene encoding the above-mentioned nicotinamide phosphoribosyltransferase mutant.

[0019] In yet another aspect, the present invention provides an expression vector comprising the above encoding gene.

[0020] Specifically, the vector includes but is not limited to: pET28a(+), pet30a(+), pETduet, pREST or pTriEX.

[0021] In certain specific embodiments of the present invention, the vector is pET28a(+).

[0022] In yet another aspect, the present invention provides a genetically engineered bacterium comprising the above-mentioned nicotinamide phosphoribosyltransferase mutant and / or the above-mentioned encoding gene and / or the above-mentioned expression vector.

[0023] Specifically, the genetically engineered bacteria include but are not limited to Escherichia coli, Salmonella, and Pichia pastoris.

[0024] In certain specific embodiments of the present invention, the engineered bacteria is Escherichia coli BL21 (DE3).

[0025] In yet another aspect, the present invention provides use of the above-mentioned nicotinamide phosphoribosyltransferase mutant and / or the genetically engineered bacteria of the above-mentioned expression vector in the preparation of β-nicotinamide mononucleotide.

[0026] In certain specific embodiments of the present invention, the genetically engineered bacteria are cultured and then centrifuged to obtain wet bacteria.

[0027] In certain specific embodiments of the present invention, the wet bacteria are ultrasonically disrupted to obtain a crude nicotinamide phosphoribosyltransferase enzyme solution.

[0028] In certain specific embodiments of the present invention, the nicotinamide phosphoribosyltransferase is purified by suspending the wet bacteria in a buffer, ultrasonically disrupting the cells, and centrifuging the cells to obtain the supernatant, and purifying the protein using a Ni affinity column.

[0029] Specifically, the genetically engineered bacteria, wet bacteria, crude enzyme solution, and nicotinamide phosphoribosyltransferase can all be used for the preparation of β-nicotinamide mononucleotide.

[0030] In yet another aspect, the present invention provides a method for preparing β-nicotinamide mononucleotide.

[0031] Specifically, the method uses the nicotinamide phosphoribosyltransferase mutant and / or the genetically engineered bacteria as a catalyst.

[0032] More specifically, the method uses ribose, nicotinamide, ATP, Tris, and magnesium chloride hexahydrate as substrates; uses ribokinase, ribose phosphate pyrophosphokinase, inorganic pyrophosphohydrolase, wild-type or mutant nicotinamide phosphoribosyltransferase as catalysts to construct a reaction system to synthesize β-nicotinamide mononucleotide in a one-step reaction.

[0033] In certain specific embodiments of the present invention, the reaction system contains 9-112.5 mM ribose, 10-125 mM nicotinamide, 20-187.5 mM ATP, 50-200 mM, 50-150 mM magnesium chloride hexahydrate; the reaction system contains 2.598-16.670 U, 86.380-540.500 U of ribose phosphate pyrophosphokinase, 78.795-497.450 U of inorganic pyrophosphohydrolase, and 5.427-55.33 U of parent or mutant nicotinamide phosphoribosyltransferase per 100 mL.

[0034] Specifically, the first reaction is carried out at 30-37°C.

[0035] In certain embodiments of the present invention, the reaction is carried out at 35°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is the reaction progress curve diagram of Example 6. DETAILED DESCRIPTION

[0037] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0038] Experimental Example 1 Transformation and induced expression of nicotinamide phosphoribosyltransferase parent and mutant

[0039] The constructed plasmid was centrifuged at 10,000 rpm for 10 min, and 50 μL ddH2O was added to dissolve it on a vortex shaker. After dissolution, it was transformed into E. coli. E. coliBL21 (DE3), and activate the transformed E. coli, place at 37ºC, 220 rpm, culture for 1 hour, spread on LB plates containing 50 µg / mL kanamycin resistance, and culture at 37 ºC inverted overnight. Pick a single colony from the plate and inoculate it into TB liquid medium containing a final concentration of 50 µg / mL kanamycin, add glucose with a final concentration of 5 g / L and 20 g / L lactose, and culture at 37 ºC, 220 rpm. When OD600 reaches 0.8-1.2 (preferably 0.9), change the temperature to 28 ºC, culture at 220 rpm for 14 hours, and centrifuge at 4 ºC, 8000 rpm / min for 10 minutes to obtain wet bacteria containing nicotinamide phosphoribosyltransferase from different sources and mutants.

[0040] Experimental Example 2 Nicotinamide phosphoribosyltransferase from different sources and screening of mutant libraries

[0041] The wet cells obtained in Experimental Example 1 were centrifuged, resuspended in pure water at a total cell volume of 40 g / L, and crushed on an ice-water mixture for 20 min. The ultrasonic crushing conditions were: 200 W, crushing for 5 s, and pausing for 7 s. After crushing, a crude enzyme solution was obtained.

[0042] The crude enzyme solution was used as a catalyst, nicotinamide and ribose phosphate pyrophosphate were used as substrates, and magnesium chloride hexahydrate was used as a cosubstrate for catalytic experiments. Since ribose phosphate pyrophosphate is expensive and unstable, ribose and disodium adenosine triphosphate were used as substrates for enzyme catalytic reaction for preparation. The reaction system was selected as 10mL, the final concentration of ribose was 2.75g / L, the final concentration of nicotinamide was 6.25g / L, the final concentration of disodium adenosine triphosphate was 25g / L, the final concentration of magnesium chloride hexahydrate was 16g / L, Tris was 24.2g / L, and the reaction pH was 8.50. First, ribose kinase 34.79U, sugar phosphate pyrophosphokinase 424.4U and inorganic pyrophosphohydrolase 89.54U were added, the reaction volume was 9mL, and the reaction was allowed to stand at 35 ºC for 1h. Then 1mL of crude enzyme solution diluted in different times was added, and 1mL of deionized water was added to the blank control group. After mixing, the reaction was allowed to stand at 35 ºC for 40min. Take 100 µL of the reaction solution, add 5 µL of hydrochloric acid to terminate the reaction. Take 10 µL of the stop solution, dilute with 1 mL of mobile phase, and filter through a 0.22 µM filter membrane as a HPLC test sample.

[0043] Liquid phase detection conditions for ATP, nicotinamide and NMN: chromatographic column Inert SustainTM C18 (4.6 × 150 mm, 5µm, Shimadzu) column, mobile phase 0.06M KH2PO4+0.007M Na2HPO4+5% methanol, flow rate 1.0 mL / min, detection wavelength 266 nm, injection volume 10 μL, column temperature 25°C.

[0044] The enzyme activity unit (U) is defined as the amount of enzyme required to produce 1 μmol of NMN in 1 min under the above conditions.

[0045] Example 1 Nampt first round screening

[0046] In this example, the bacteria derived from Thermus rubrum ( Meiothermus ruber DSM1279 ) was used as a template to screen for nicotinamide phosphoribosyltransferases with high catalytic activity.

[0047] Eight amino acid sequences were screened from the NCBI database. The nicotinamide phosphoribosyltransferases corresponding to these eight amino acid sequences were denoted as Nampt[Ml], Nampt[Dp], Nampt[Dc], Nampt[Tp], Nampt[Ap], Nampt[Fd], Nampt[Pf] and Nampt[Fl], respectively. The Nampt[Mr] enzyme was Meiothermus ruber DSM 1279 Based on the nucleotide sequence of 2889129-2890520 of the genome (GenBank: CP001743.1), Nampt[Ml] enzyme Meiothermus luteus strain KCTC 52599 contig_33 Based on the 8309-9700 nucleotide sequence of the genome (GenBank: QWKZ01000033.1), Nampt[Dp] enzyme Deinococcus peraridilitoris DSM 19664 Based on the nucleotide sequence of 2545493-2546905 of the genome (GenBank: CP003382.1), Nampt[Dc] enzyme was Deinococcus cellulosilyticus NBRC 106333 = KACC 11606 DNA, sequence08 Based on the nucleotide sequence of 18095-19468 of the genome (GenBank: BJXB01000008.1), Nampt[Tp] enzyme Trichinella papuae isolate ISS1980 scaffold125s The Nampt[Ap] enzyme was based on the nucleotide sequence collection of 42651~42906,42975~43105, 43210~43368, 43979~44107, 44171~44404, 44456~44500 of the genome (GenBank: JYDO01000125.1). Acanthochromis polyacanthus nicotinamide phosphoribosyltransferase 2 Based on the nucleotide sequence of 88-1569 of mRNA of (nampt2) (GenBank: XM_022219777.2), Nampt[Fd] enzyme was Fructobacillus durionis strain DSM 19113 genome assembly, contig: Ga0056052_102Based on the nucleotide sequence of 114933~116345 of the genome (GenBank: FOLI01000002.1), the Nampt[Pf] enzyme was Paenibacillus ferrarius strain CY1 CY1_ scaffold56 Based on the 2876-4363 nucleotide sequence of the genome (GenBank: NZ_MBTG01000052.1), the Nampt[Fl] enzyme was Flavobacterium sp. Leaf359 contig_10 Based on the nucleotide sequence 22944-24395 of the genome (GenBank: LMPW01000002.1), the homologies of these amino acid sequences with the template amino acid sequence were 91.1%, 58.9%, 53.9%, 48.9%, 43.5%, 38.5%, 33.3% and 28.4%, respectively.

[0048] according to E.coli Codon preference was used to optimize the rare codons in these nucleotide sequences, add an NcoⅠ restriction site before the start codon (CCATGG, if the second codon starts with G, only CC is added, if the second codon does not start with G, CC is added before ATG, and GGC is added after ATG, that is, an extra glycine is added), delete the stop codon, and add an XhoⅠ restriction site at the end of the sequence; these sequences were sent to Shanghai Jierui for whole gene synthesis, and the NcoⅠ / XhoⅠ double restriction site was used to connect to the pET28a(+) vector to construct p ET28(+)-Nampt[Mr], pET28(+)-Nampt[Ml], pET28(+)-Nampt[Dp], pET28(+)-Nampt[Dc], pET28(+)-Nampt[Tp], pET28(+)-Nampt[Ap], pET28(+)-Nampt[Fd], pET28(+)-Nampt[Pf] and pET28(+)-Nampt[Fl], the activity of all enzymes was detected according to the methods of Experimental Examples 1 and 2.

[0049] The experimental results are shown in Table 1. It can be seen from Table 1 that the activities of Nampt[Dc] and Nampt[Fd] are higher than those of Nampt[Mr], and the activities of other enzymes are lower than those of Nampt[Mr]. Among them, Nampt[Dc] has the highest catalytic activity, which is 564.59% of Nampt[Mr], and the sequence homology is 53.9%. In addition, the experiment shows that there is no necessary connection between the catalytic activity of the screened nicotinamide phosphoribosyltransferase and the sequence homology with the template.

[0050] Table 1

[0051]

[0052] Example 2 Nampt Second Round Screening

[0053] In this example, Nampt[Dc] with the highest activity in Example 1 was used as a template to screen for nicotinamide phosphoribosyltransferase with high catalytic activity.

[0054] Eight amino acid sequences were screened from the NCBI database, and Nampt[Di] enzyme was Deinococcus irradiatisoli strain 17bor-2 chromosome Based on the nucleotide sequence of 2452426-2453832 of the genome (GenBank: NZ_CP029494.1), Nampt[Ss] enzyme Synechococcus sp. PCC 6312 Based on the nucleotide sequence of 3391322~3392695 of the genome (GenBank: CP003558.1), Nampt[Gk] enzyme was Gloeobacter kilaueensis JS1 Based on the nucleotide sequence of 3407929-3409308 of the genome (GenBank: CP003587.1), Nampt[Di] enzyme was Deinococcus irradiatisoli strain 17bor-2 chromosome Based on the nucleotide sequence of 2452426-2453832 of the genome (GenBank: NZ_CP029494.1), the Nampt[Ms] enzyme was Microbulbifer sp. Based on the nucleotide sequence 4552708-4554123 of A4B17 chromosome genome (GenBank: NZ_CP029064.1), Nampt[Pa] enzyme was Pseudobacteriovorax antillogorgiicola strain DSM 103413 Ga0244732_104 genome (GenBank: SLZT01000004.1), the Nampt[Ar] enzyme was expressed as Acipenser ruthenus isolate WHYD16114868_AA scaffold312 Genome (GenBank: SCEB01214929.1): 237438~237578, 238485~238676, 239358~239562, 240269~240387, 244001~244072,248729~248788, 249071~249205, 254635~254775, 256108~256227, 257979~258204,260019~260155, 260343~260501, 261760~261888, 262779~262874, Based on the nucleotide sequence collection of 269690~269743, Nampt[Rm] enzyme is Rhizosphaericola mali strain B3-10 chromosomeThe nucleotide sequences of 4471612 to 4473069 of the genome (GenBank: CP044016.1) were used as the basis; the homologies of these amino acid sequences with the template amino acid sequence were 65.4%, 62.5%, 59.2%, 54.4%, 49.2%, 44.0%, 38.8% and 34.4%, respectively.

[0055] According to the codon preference of E. coli, the rare codons in these nucleotide sequences were optimized, and an NcoⅠ restriction site was added before the start codon (CCATGG, if the second codon starts with G, only CC is added, if the second codon does not start with G, CC is added before ATG, and GGC is added after ATG, that is, an extra glycine is added), the stop codon was deleted, and an XhoⅠ restriction site was added to the end of the sequence; these sequences were sent to Shanghai Jierui for whole gene synthesis, and ligated to p The ET28a(+) vector was used to construct pET28(+)-Nampt[Di], pET28(+)-Nampt[Ss], pET28(+)-Nampt[Gk], pET28(+)-Nampt[Ms], pET28(+)-Nampt[Pa], pET28(+)-Nampt[Ar], pET28(+)-Nampt[Cv] and pET28(+)-Nampt[Rm], and the activities of all enzymes were detected according to Experimental Examples 1 and 2.

[0056] The experimental results are shown in Table 2. The enzyme activities of these 8 enzymes are all lower than that of Nampt[Dc].

[0057] Table 2

[0058]

[0059] Example 3 Mutation of Nampt[Dc]

[0060] Saturation mutagenesis of F180, E231, D298, D338 and D377 of Nampt[Mr] revealed that the two sites E231 and D338 were more likely to obtain mutants with higher activity. Multiple sequence alignment revealed that the two amino acids E231 and D338 of Nampt[Mr] were very conserved, corresponding to the E227 and D334 sites of Nampt[Dc], so saturation mutagenesis was performed on these two sites.

[0061] The D334 of Nampt[Dc] was subjected to site-directed saturation mutagenesis, and the point mutation was sent to Shanghai Jierui for completion, obtaining the recombinant plasmids pET28(+)-Nampt[Dc]D334V, pET28(+)-Nampt[Dc] D334Y, pET28(+)-Nampt[Dc]D334I, pET28(+)-Nampt[Dc] D334M, pET28(+)-Nampt[Dc]D334K, pET28(+)-Nampt[Dc] D334E, pET28(+)-Nampt[Dc]D334C, pET28(+)-Nampt[Dc] D334T, pET28(+)-Nampt[Dc]D334R, and pET28(+)-Nampt[Dc] The enzyme activity was detected according to the method of Experimental Examples 1 and 2.

[0062] The test results are shown in Table 3. The six forward mutants, namely D334Y, D334M, D334S, D334A, D334F and D334N, had enzyme activities that were 112.86%, 148.62%, 117.99%, 132.04%, 116.71% and 114.33% of the wild type, respectively.

[0063] Table 3

[0064] Mutant Vitality (%) Mutant Vitality (%) Mutant Vitality (%) Nampt[Dc] WT 100 Nampt[Dc] D334V 92.41 Nampt[Dc] D334Y 112.86 Nampt[Dc] D334I 80.59 Nampt[Dc] D334M 148.62 Nampt[Dc] D334K 70.83 Nampt[Dc] D334E 69.47 Nampt[Dc] D334C 30.78 Nampt[Dc] D334T 104.00 Nampt[Dc] D334R 19.86 Nampt[Dc] D334S 117.99 Nampt[Dc] D334G 76.31 Nampt[Dc] D334A 132.04 Nampt[Dc] D334P 1.87 Nampt[Dc] D334W 66.77 Nampt[Dc] D334Q 16.77 Nampt[Dc] D334F 116.71 Nampt[Dc] D334H 86.92 Nampt[Dc] D334L 43.21 Nampt[Dc] D334N 114.33 - -

[0065] The site-directed saturation mutagenesis of E227 of Nampt[Dc] was sent to Shanghai Jierui for completion, and the recombinant plasmids pET28(+)-Nampt[Dc]E227V, pET28(+)-Nampt[Dc] E227Y, pET28(+)-Nampt[Dc]E227I, pET28(+)-Nampt[Dc] E227M, pET28(+)-Nampt[Dc]E227K, pET28(+)-Nampt[Dc] E227D, pET28(+)-Nampt[Dc]E227C, pET28(+)-Nampt[Dc] E227T, pET28(+)-Nampt[Dc]E227R, and pET28(+)-Nampt[Dc] E227S, pET28(+)-Nampt[Dc]E227G, pET28(+)-Nampt[Dc] E227A, pET28(+)-Nampt[Dc]E227P, pET28(+)-Nampt[Dc] E227W, pET28(+)-Nampt[Dc]E227Q, pET28(+)-Nampt[Dc] E227F, pET28(+)-Nampt[Dc]E227H, pET28(+)-Nampt[Dc] E227L and pET28(+)-Nampt[Dc]E227N, the enzyme activity was detected according to the methods of Experimental Examples 1 and 2.

[0066] The test results are shown in Table 4. The saturation mutation obtained four positive mutants, namely E227S, E227N, E227Q and E227A, and the enzyme activities were 128.71%, 120.77%, 119.52%, and 119.89% of the wild type, respectively.

[0067] Table 4

[0068]

[0069] The forward mutants obtained from D334 and E227 were subjected to two-by-two combined mutations, and the point mutations were sent to Shanghai Jierui for completion to obtain the recombinant plasmids pET28(+)-Nampt[Dc] E227AD334Y, pET28(+)-Nampt[Dc]E227SD334Y, pET28(+)-Nampt[Dc] E227QD334Y, pET28(+)-Nampt[Dc]E227ND334Y, pET28(+)-Nampt[Dc]E227AD334M, pET28(+)-Nampt[Dc]E227SD334M, and pET28(+)-Nampt[Dc] E227QD334M, pET28(+)-Nampt[Dc]E227ND334M, pET28(+)-Nampt[Dc] E227AD334S, pET28(+)-Nampt[Dc]E227SD334S, pET28(+)-Nampt[Dc] E227QD334S, pET28(+)-Nampt[Dc]E227ND334S, pET28(+)-Nampt[Dc] E227AD334A, pET28(+)-Nampt[Dc]E227SD334A, pET28(+)-Nampt[Dc]E227QD334A, pET28(+)-Nampt[Dc]E227ND334A, pET28(+)-Nampt[Dc] E227AD334F, pET28(+)-Nampt[Dc]E227SD334F, pET28(+)-Nampt[Dc] E227QD334F, pET28(+)-Nampt[Dc]E227ND334F, pET28(+)-Nampt[Dc] E227AD334N, pET28(+)-Nampt[Dc]E227SD334N, pET28(+)-Nampt[Dc] E227QD334N and pET28(+)-Nampt[Dc]E227ND334N, the enzyme activity was detected according to the method of Experimental Examples 1 and 2.

[0070] The experimental results are shown in Table 5. There are 4 reverse mutants whose enzyme activities are lower than that of the wild-type enzyme, and the enzyme activities of the remaining mutants are higher than that of the wild-type enzyme. Among them, the higher ones are Nampt[Dc] E227AD334M (176.19%), Nampt[Dc]E227ND334M (190.01%), Nampt[Dc] E227QD334S (163.24%), Nampt[Dc]E227QD334A (172.35%) and Nampt[Dc] E227SD334N (150.03%).

[0071] Table 5

[0072] Mutant child vitality (%) Mutant child vitality (%) Mutant child vitality (%) Nampt[Dc] WT 100.00 Nampt[Dc] E227AD334Y 140.05 Nampt[Dc] E227SD334Y 110.41 Nampt[Dc] E227QD334Y 104.33 Nampt[Dc] E227ND334Y 131.42 Nampt[Dc] E227AD334M 176.19 Nampt[Dc] E227SD334M 139.61 Nampt[Dc] E227QD334M 138.48 Nampt[Dc] E227ND334M 190.01 Nampt[Dc] E227AD334S 113.22 Nampt[Dc] E227SD334S 89.15 Nampt[Dc] E227QD334S 163.24 Nampt[Dc] E227ND334S 93.22 Nampt[Dc] E227AD334A 135.23 Nampt[Dc] E227SD334A 143.20 Nampt[Dc] E227QD334A 172.35 Nampt[Dc] E227ND334A 131.20 Nampt[Dc] E227AD334F 118.53 Nampt[Dc] E227SD334F 110.42 Nampt[Dc] E227QD334F 106.78 Nampt[Dc] E227ND334F 126.65 Nampt[Dc] E227AD334N 120.13 Nampt[Dc] E227SD334N 150.03 Nampt[Dc] E227QD334N 66.43 Nampt[Dc] E227ND334N 78.99 - - - -

[0073] Example 4: Purification of Nicotinamide Phosphoribosyltransferase and Its Mutants

[0074] First, the wet cells of the Nampt[Dc] parent and dominant mutants (in Table 5) obtained in Example 3 were suspended in buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole), ultrasonically disrupted for 15 min (ice bath, power 400 W, disruption for 3 seconds, pause for 5 seconds), centrifuged at 4°C and 12,000 rpm for 20 min, and the supernatant was collected. The protein was purified using a Ni affinity column (1.6×10 cm, Bio-Rad, USA) with the following steps: ① Equilibrate the Ni column with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl and 20 mM imidazole) until the baseline was stable; ② Load the sample at a flow rate of 1 mL / min with a loading amount of 25-40 mg / mL protein to allow the target protein to adsorb on the Ni column; ③ Wash the impurities with 6 column volumes of buffer A (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl, 20 mM imidazole) at a flow rate of 1 mL / min until the baseline was stable; ④ Elute with buffer B (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl, 400 mM imidazole) at a flow rate of 1 mL / min to collect the target protein. The target protein was dialyzed overnight in pH 8.0, 20 mM Tris buffer to obtain purified nicotinamide phosphoribosyltransferase; ⑤ The Ni column was rinsed with 5 column volumes of binding buffer (pH 8.0, 20 mM Tris buffer containing 0.5 M NaCl) until the baseline was stable, and the Ni column was preserved with 5 column volumes of ultrapure water containing 20% ​​ethanol.

[0075] Example 5: Preparation of low concentration nicotinamide mononucleotide using nicotinamide phosphoribosyltransferase and its mutants

[0076] According to the description of Example 4, the purified pure enzyme solution was taken, six experimental groups were set up, and two parallel samples were set up in each experimental group. The reaction base solution was prepared, 9mM ribose, 10mM nicotinamide, 20mM ATP, 50mM Tris, 50mM magnesium chloride hexahydrate, and hydrochloric acid was added to adjust the pH to 8.5. 2.598U of ribokinase, 86.380U of sugar phosphate pyrophosphokinase, 78.795U of inorganic pyrophosphohydrolase, and 5.427U of parent or mutant nicotinamide phosphoribosyltransferase were added. The reaction was allowed to stand at 35°C. The generation of the product NMN during the reaction was detected by the liquid phase method shown in Example 5. After 5-6h of reaction, the conversion rate of NMN reached more than 80%, and the highest could reach 82%, and the yield of NMN was 2.67g / L.

[0077] Example 6: Preparation of high concentration nicotinamide mononucleotide using nicotinamide phosphoribosyltransferase and its mutants

[0078] According to the data, ADP has an inhibitory effect on the activity of sugar phosphate pyrophosphokinase. In order to reduce the inhibitory effect, adenylate kinase was added to the reaction system.

[0079] According to the description in Example 5, take the purified pure enzyme solution, the reaction system is 100 mL, set up six experimental groups, and set up two parallel samples in each experimental group. Prepare the reaction base solution, 112.5mM ribose, 125mM nicotinamide, 187.5mM ATP, 200mM Tris, 150mM magnesium chloride hexahydrate, add hydrochloric acid to adjust the pH to 8.5, and set aside. Add 16.67U of ribokinase, 540.50U of sugar phosphate pyrophosphokinase, 497.45U of inorganic pyrophosphohydrolase, 55.33U of parent or mutant nicotinamide phosphoribosyltransferase, and 800U of adenylate kinase. Let the reaction stand at 35°C. The formation of the product NMN during the reaction was detected by the liquid phase method shown in Example 5. The reaction progress curve is shown in Figure 1 After 20 hours of reaction, the conversion rate of NMN reached more than 70%, with the highest reaching 71%, and the yield of NMN was 29.24 g / L.

Claims

1. A nicotinamide phosphoribosyltransferase mutant, characterized in that: The nicotinamide phosphoribosyltransferase mutant is obtained by performing a single point mutation or a double point mutation at position 227 and / or position 334 of the nicotinamide phosphoribosyltransferase shown in SEQ ID NO: 1; The single point mutation at position 227 is one of E227S, E227N, E227Q or E227A; The single point mutation at position 334 is one of D334Y, D334M, D334S, D334A, D334F or D334N; The double point mutation at position 227 and position 334 is one of: E227A and D334M, E227N and D334M, E227Q and D334S, E227Q and D334A or E227S and D334N.

2. A gene encoding the nicotinamide phosphoribosyltransferase mutant according to claim 1.

3. An expression vector comprising the coding gene according to claim 2.

4. The expression vector according to claim 3, characterized in that The expression vector was constructed based on pET28a(+).

5. A genetically engineered bacterium comprising the nicotinamide phosphoribosyltransferase mutant according to any one of claim 1 and / or the encoding gene according to claim 2 and / or the expression vector according to any one of claims 3-4.

6. The genetically engineered bacterium according to claim 5, characterized in that: The genetically engineered bacteria is Escherichia coli BL21 (DE3).

7. Use of the nicotinamide phosphoribosyltransferase mutant according to claim 1 and / or the encoding gene according to claim 2 and / or the expression vector according to any one of claims 3-4 and / or the genetically engineered bacteria according to any one of claims 5-6 in the preparation of β-nicotinamide mononucleotide.

8. A method for preparing β-nicotinamide mononucleotide, characterized in that: The method uses the nicotinamide phosphoribosyltransferase mutant according to claim 1 and / or the genetically engineered bacteria according to any one of claims 5-6 as a catalyst.

9. The method according to claim 8, characterized in that The method uses ribose, nicotinamide, ATP, Tris, and magnesium chloride hexahydrate as substrates; uses ribokinase, ribose phosphate pyrophosphokinase, inorganic pyrophosphohydrolase, and mutant nicotinamide phosphoribosyltransferase as catalysts to construct a reaction system and synthesize β-nicotinamide mononucleotide by a one-step reaction.

10. The method according to claim 9, characterized in that The reaction system contains 9-112.5 mM ribose, 10-125 mM nicotinamide, 20-187.5 mM ATP, 50-200 mM, 50-150 mM magnesium chloride hexahydrate.

11. The method according to claim 10, characterized in that The reaction system contains 2.598-16.670U, 86.380-540.500U of ribose phosphate pyrophosphokinase, 78.795-497.450U of inorganic pyrophosphohydrolase, and 5.427-55.33U of mutant nicotinamide phosphoribosyltransferase per 100mL.

12. The method according to claim 11, characterized in that The one-step reaction is carried out at 30-37°C.

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