Mutant of nicotinamide adenine dinucleotide synthetase and application thereof

Abstract

The invention relates to the technical field of biological enzyme engineering, in particular to a mutant of nicotinamide adenine dinucleotide synthetase and application thereof. According to the nicotinamide adenine dinucleotide synthetase mutant, mutation of sites such as W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E and L133T is carried out by taking NadR as a parent, so that the mutant can efficiently catalyze a low-cost substrate NR and ATP to generate NAD. Especially through mutation of the multiple mutation sites, the obtained mutant has better substrate binding capacity and catalytic efficiency and higher NAD conversion rate and enzyme activity (up to 12.49 U / mg), not only solves the technical problem of low activity of the existing NAD synthetase, but also remarkably reduces the production cost of NAD by using low-price NR instead of expensive NMN as a substrate, and has good application prospects. And a technical scheme with more economic benefits is provided for industrial production of NAD.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological enzyme engineering, and particularly relates to a mutant of nicotinamide adenine dinucleotide synthetase and application thereof. BACKGROUND

[0002] At present, the industrial production of NAD mainly adopts two ways of chemical method and biological enzyme catalysis method. The biological enzyme catalysis method has gradually become the mainstream method for producing NAD due to the advantages of mild reaction conditions, energy saving and environmental protection, and no organic solvent residues. In the prior art, the biological enzyme catalysis method of NAD is mainly to synthesize NAD from nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) under the catalysis of nicotinamide mononucleotide adenosine transferase (NMNAT). However, the substrate NMN used in this method is expensive, which leads to high production cost of NAD and lack of advantages in market competition.

[0003] Although NAD synthetase can catalyze the one-step generation of NAD from nicotinamide riboside (NR) and ATP with a lower price, the existing NAD synthetase has generally low activity and is difficult to meet the needs of industrial production.

[0004] Therefore, it is urgent to develop a mutant of NAD synthetase with high catalytic activity to realize the production of NAD by replacing the high-cost NMN with the low-cost substrate NR, so as to reduce the production cost of NAD and improve the market competitiveness of the product. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application provides a mutant of nicotinamide adenine dinucleotide synthetase and application thereof. The mutant can efficiently catalyze the synthesis of NAD from low-cost substrate NR and ATP, significantly improve the catalytic activity of the enzyme, and greatly improve the yield of NAD.

[0006] A mutant of nicotinamide adenine dinucleotide synthetase, which is obtained by mutating a gene editing the amino acid sequence of drNadR shown in SEQ ID NO. 1, and comprises any one of the following mutation sites: W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, and L133T.

[0007] Further, the amino acid sequence of the mutant of nicotinamide adenine dinucleotide synthetase is shown in any one of SEQ ID NO. 77 to SEQ ID NO. 86.

[0008] The present application has the following advantages: The nicotinamide adenine dinucleotide synthetase mutant provided by the present application can efficiently catalyze the reaction of low-cost substrate NR and ATP to generate NAD by mutating the W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, L133T sites of NadR as the parent. Especially by the above-mentioned multi-mutation point mutation, the mutant has better substrate binding capacity and catalytic efficiency, and higher NAD conversion rate and enzyme activity (up to 12.49 U / mg). Not only does it solve the technical problem of low activity of existing NAD synthetase, but also significantly reduces the production cost of NAD by using low-cost NR instead of expensive NMN as the substrate, providing a more economically viable technical solution for the industrial production of NAD. BRIEF DESCRIPTION OF DRAWINGS

[0009] The present application will be further described below in conjunction with the drawings and examples.

[0010] Figure 1 is the liquid chromatogram of the reaction result of Example 8; Figure 2 is the liquid chromatogram of the standard; Figure 3 is Figure 1 and Figure 2 is the comparison chart of and. DETAILED DESCRIPTION

[0011] As used herein, "and / or" includes the term "and" and the term "or", and any other combination of the two terms. The terms used herein are used only to describe particular embodiments, and are not intended to limit the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms, unless the context clearly indicates otherwise. It is further understood that "comprising" as used in the specification, specifies the stated features, integers, steps, operations, elements, and / or components, but does not exclude one or more additional features, integers, steps, operations, elements, components, and / or groups thereof.

[0012] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0013] The exemplary applications described herein can appropriately lack any one or more of the element limitations not specifically disclosed herein. Thus, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms "comprising," "having," "including," and the like can be used interchangeably in the present disclosure. Furthermore, the described features, advantages, and characteristics of the application can be combined in any suitable manner to form variations of the application. Accordingly, other implementations of or of two or more additional implementations of the application not expressly disclosed herein are also possible and contemplated. Thus, the scope of the application should be determined by the appended claims and their legal equivalents, rather than by the description of the application. It will be understood that, when an element or layer is referred to as being "on" or "connected to" another element or layer, it can be directly on or connected to the other element or layer or intervening elements or layers can be present. In contrast, when an element is referred to as being "directly on" or "directly connected to" another element, there are no intervening elements or layers present. It will also be understood that, when an element is referred to as being "coupled" or "connected" to another element, it can be electrically, mechanically or communicatively coupled or connected to the other element unless expressly stated otherwise. In contrast, when an element is referred to as being "directly coupled" or "directly connected" to another element, there are no intervening elements or layers present. It will be understood that the terms "coupled" or "connected" can refer to an electrical connection, a mechanical connection, an optical connection, a magnetic connection, a physical connection, or any combination thereof.

[0014] The raw materials or reagents used in the examples and comparative examples of the present application are purchased from mainstream manufacturers in the market. If the manufacturer is not specified or the concentration is not specified, it is an analytical pure raw material or reagent that can be obtained conventionally, without particular limitation, as long as it can play the expected role. The reaction, stirring and other instruments and equipment used in the examples are purchased from major manufacturers in the market, without particular limitation, as long as they can play the expected role. If the specific technology or condition is not specified in the examples, it is carried out according to the technology or condition described in the literature in the art or according to the product instruction.

[0015] The present application first proposes a mutant of nicotinamide adenine dinucleotide synthetase. By site-directed mutagenesis on the amino acid sequence of zebrafish NadR, mutations are introduced at specific sites (such as W11F, M12R, etc.), and the mutant is obtained by genetic engineering means. The mutant has higher catalytic activity and can improve the synthesis efficiency of NMN.

[0016] Example 1 The amino acid sequence of zebrafish NadR (drNadR) is shown in SEQ ID NO. 1.

[0017] The preparation method of the mutant of nicotinamide adenine dinucleotide synthetase comprises: S1. Constructing an original recombinant plasmid: (1) obtaining the amino acid sequence corresponding to the NadR gene from Danio rerio; (2) codon optimization and synthesis of the gene sequence of the amino acid sequence; (3) constructing the gene sequence into a pET22b(+) vector to obtain a recombinant plasmid pET22b(+)-drNadR; S2. Determining the mutation site: The substrate, product and three-dimensional structure of the drNadR amino acid sequence are analyzed by molecular docking method to determine the mutation site in the active pocket; S3. Site-directed mutagenesis: The recombinant plasmid is subjected to site-directed mutation, and the mutant recombinant plasmid is obtained after DpnI enzyme digestion treatment; S4. Obtain the mutant: The mutant recombinant plasmid is transformed into an expression strain, cultured and induced to express to obtain the mutant.

[0018] In step S1, the amino acid sequence corresponding to the NadR gene derived from Danio rerio (Genebank: NP_001004618) is screened out in the NCBI library, and codon optimization and gene sequence synthesis are performed in Universal Biological (Anhui) Co., Ltd. to obtain the recombinant plasmid pET22b (+) -drNadR. Then, the obtained recombinant plasmid is transformed into DH5a competent cells, single colonies are selected for colony PCR screening, positive single colony strains are selected for preservation and plasmid extraction, and the amino acid sequence is confirmed to be correct by sequencing. The positive recombinant plasmid pET22b-drNadR is transformed into the expression host strain BL21 (DE3) to obtain the expression strain drNadR.

[0019] In step S2, the three-dimensional structures of the substrate NR, the product NAD and the drNadR amino acid sequence are subjected to molecular docking by AUTODOCK, the active pocket of the amino acid sequence is determined according to the preliminary results, and the first round of saturation mutation is determined after screening. The mutation sites are W11, M12, H37, I38, I40, E42, G43, F44, L46, Y47, V55, N57, C59, F60, Y61, V62, L112, L125, I129, and L133. KOD OneTM PCR Master Mix of Toyo Bao is used for whole plasmid site-directed mutation PCR; after the PCR reaction, whole enzyme DMT Enzyme (DpnI) is used for digestion, and after the digestion is completed, it is directly transformed into the DH5a strain, single colonies are selected and sequenced.

[0020] In step S3, the site-directed mutation includes at least one of the following sites: W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, and L133T. When the site-directed mutation is a single point mutation, the primers used are shown in SEQ ID NO. 2-41.

[0021] Example 2 Using a similar method to Example 1, a comparative plasmid is constructed: 1. Amplifying NadR gene from E. coli genome by primer PCR and adding restriction sites NdeI and XhoI at both ends respectively; The primer sequences are as follows: ecNadR-F: 5'-GGAATTCCATATGTCGTCATTTGATTAC-3' and ecNadR-R: 5'- CCGCTCGAGTTGTCTCTGCTCCCCCATC-3'; 2. Obtaining recombinant plasmid pET22b (+) -ecNadR by enzyme digestion and ligation of E. coli NadR gene and plasmid pET22b (+); 3. Transforming the obtained recombinant plasmid pET22b (+) -ecNadR into DH5a competent cells, selecting single colonies for colony PCR screening, selecting positive single colony strains for preservation and plasmid extraction, and confirming the correct amino acid sequence by sequencing.

[0022] Transforming the positive recombinant plasmid pET22b (+) -ecNadR into expression host bacteria BL21 (DE3) to obtain expression strain ecNadR, wherein the amino acid sequence of ecNadR is shown as SEQ ID NO. 42.

[0023] Example 3 The enzyme activities of ecNadR, drNadR and each mutant thereof were determined by the above method.

[0024] It should be noted that the enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 micromole of nicotinamide riboside into nicotinamide adenine dinucleotide in 1 minute at 37°C.

[0025] Test method: Dilute the enzyme solution to 1 mg / mL, and add 100 μL of enzyme solution to 100 μL of reaction solution (final concentration of pH 6.0 100 mM potassium dihydrogen phosphate buffer, 60 mM magnesium chloride, 120 mM nicotinamide riboside and 120 mM adenosine triphosphate), and react at 37°C for 5 min.

[0026] After the reaction is completed, the molar concentration of nicotinamide adenine dinucleotide NAD in the reaction solution is determined by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0027] Table 1

[0028] As can be seen from the data in Table 1, the drNadR mutants after single-point mutation modification all show significantly improved catalytic activity. Among them, the NAD production (7.01-8.62 mM) and enzyme activity (2.80-3.45 U / mg) of all mutants are significantly higher than those of the original ecNadR (0.01 mM, 0.01 U / mg) and drNadR (5.11 mM, 2.04 U / mg).

[0029] Example 4 The site-directed mutation is a multi-point mutation, and the primers used are shown in SEQ ID NO. 43-76. The plasmid of drNadR is used as a template, and the primers are designed to perform combined mutations of W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, L133T, wherein the results of the ten strains with the highest enzyme activity are shown in Table 2, and the amino acid sequences are shown in SEQ ID NO. 77-86.

[0030] Table 2

[0031] Example 5 Enzyme activity of mutant drNadR-M12R / H37W / I40L / L46E / V55E / L112I / L125E: 34.92 g nicotinamide riboside, 66.14 g adenosine triphosphate, 12.2 g magnesium chloride, 9.7 g potassium phosphate dibasic, 1.02 g potassium phosphate monobasic were dissolved with 646 ml water, and the pH was adjusted to 6.0. Then 354 ml of enzyme solution was added, and placed in a 37°C water bath with 200 rpm mechanical stirring for 5 minutes.

[0032] Result: 41.3 mM nicotinamide adenine dinucleotide was generated, and the enzyme activity of the mutant protein = 23.33 U / mg.

[0033] Example 6 Enzyme activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: 34.92 g nicotinamide riboside, 66.14 g adenosine triphosphate, 12.2 g magnesium chloride, 9.7 g potassium phosphate dibasic, 1.02 g potassium phosphate monobasic were dissolved with 646 ml water, and the pH was adjusted to 6.0. Then 354 ml of enzyme solution was added, and placed in a 37°C water bath with 200 rpm mechanical stirring for 5 minutes.

[0034] Result: 52.6 mM nicotinamide adenine dinucleotide was generated, and the enzyme activity of the mutant protein = 29.72 U / mg.

[0035] Example 7 Enzymatic activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: 34.92 g of nicotinamide riboside, 66.14 g of adenosine triphosphate, 12.2 g of magnesium chloride, 9.7 g of potassium phosphate dibasic, 1.02 g of potassium phosphate monobasic were dissolved with 646 ml of water, pH was adjusted to 5.5. 354 ml of enzyme solution was added, placed in a 37°C water bath, mechanically stirred at 200 rpm for 5 minutes.

[0036] Result 62.4 mM of nicotinamide adenine dinucleotide was generated, the enzyme activity of this mutant protein = 35.25 U / mg.

[0037] Example 8 Enzymatic activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: 34.92 g of nicotinamide riboside, 66.14 g of adenosine triphosphate, 12.2 g of magnesium chloride, 9.7 g of potassium phosphate dibasic, 1.02 g of potassium phosphate monobasic, 3.2 g of ammonium chloride were dissolved with 646 ml of water, pH was adjusted to 5.5. 354 ml of enzyme solution was added, placed in a 37°C water bath, mechanically stirred at 200 rpm for 5 minutes.

[0038] Result 72.3 mM of nicotinamide adenine dinucleotide was generated, the enzyme activity of this mutant protein = 40.85 U / mg.

[0039] Example 9 Enzymatic activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: 58.2 g of nicotinamide riboside, 66.14 g of adenosine triphosphate, 12.2 g of magnesium chloride, 9.7 g of potassium phosphate dibasic, 1.02 g of potassium phosphate monobasic, 3.2 g of ammonium chloride were dissolved with 820 ml of water, pH was adjusted to 5.5. 180 ml of enzyme solution was added, placed in a 37°C water bath, mechanically stirred at 200 rpm for 5 minutes.

[0040] Result 99.26 mM of nicotinamide adenine dinucleotide was generated, the enzyme activity of this mutant protein = 110.29 U / mg.

[0041] Example 10 Enzymatic activity of control strain ecNadR: 34.92 g of nicotinamide riboside, 66.14 g of adenosine triphosphate, 12.2 g of magnesium chloride, 9.7 g of potassium phosphate dibasic, 1.02 g of potassium phosphate monobasic, 3.2 g of ammonium chloride were dissolved with 646 ml of water, pH was adjusted to 5.5. 354 ml of enzyme solution was added, placed in a 37°C water bath, mechanically stirred at 200 rpm for 5 minutes.

[0042] The result is 0.32 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein = 0.18 U / mg.

[0043] Example 11 The enzyme activity of the control strain ecNadR: 58.2 g of nicotinamide riboside, 66.14 g of adenosine triphosphate, 12.2 g of magnesium chloride, 9.7 g of dipotassium hydrogen phosphate, 1.02 g of potassium dihydrogen phosphate, and 3.2 g of ammonium chloride were dissolved in 820 ml of water, and the pH was adjusted to 5.5. Then, 180 ml of enzyme solution was added, and the mixture was placed in a 37°C water bath and mechanically stirred at 200 rpm for 5 minutes.

[0044] The result is 0.40 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein = 0.44 U / mg.

[0045] Among them, examples 5 and 6: using the two strains with the highest enzyme activity after mutation, under the same reaction conditions, the enzyme activity of the two strains is compared; Example 6 and 7: adjust the pH value in the reaction system, reduce the pH value from 6.0 to 5.5, and increase the enzyme activity from 29.72 U / mg to 35.25 U / mg; Example 7 and 8: increase the amount of ammonium chloride in the enzyme catalytic system, and increase the enzyme activity from 35.25 U / mg to 40.85 U / mg; Example 8 and 9: increase the concentration of substrate nicotinamide riboside (NR) and reduce the amount of enzyme, and the enzyme activity increases to 110.29 U / mg; Example 8 and 10: compare the enzyme activity of drNadR mutant and ecNadR under the same reaction conditions, and the enzyme activity of drNadR mutant is much higher than that of ecNadR; Example 9 and 11: compare the enzyme activity of drNadR mutant and ecNadR under the same reaction conditions, and the enzyme activity of drNadR mutant is much higher than that of ecNadR.

[0046] As can be seen from examples 3-11, the strain with multiple site combination mutation significantly improves the catalytic performance of drNadR, and the effect is more significant than that of single point mutation.

[0047] Among them, include: M12R / H37W / I40L / G43D / V55E / L112I / L125E, The two seven-site mutation combinations of M12R / H37W / I40L / L46E / V55E / L112I / L125E performed the best, with NAD production reaching 31.22 mM and 30.48 mM, respectively, and enzyme activity reaching 12.49 U / mg and 12.19 U / mg, respectively.

[0048] The above values are significantly higher than those of the original enzyme and single-site mutants. This indicates that the combined mutation of these key sites has a synergistic effect, successfully achieving significant optimization of enzyme activity.

[0049] SEQ ID NO. 1 MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIHILIVEGFLLYNYKPLIDVYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQLYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 2 11F: GAATACCGTTAAAGGCNNKATGGAAAATCCGGTTA; SEQ ID NO. 3 11R: TAACCGGATTTTCCATMNNGCCTTTAACGGTATTC; SEQ ID NO. 4 12F: TACCGTTAAAGGCTGGNNKGAAAATCCGGTTAA; SEQ ID NO. 5 12R: TTAACCGGATTTTCMNNCCAGCCTTTAACGGTA; SEQ ID NO. 6 37F: CGATCCGGATAGTGATATTNNKATTCTGATTGTTG; SEQ ID NO. 7 37R: CAACAATCAGAATMNNAATATCACTATCCGGATCG; SEQ ID NO. 8 38F: TCCGGATAGTGATNNKCATATTCTGATTGTTGAGG; SEQ ID NO. 9 38R: CCTCAACAATCAGAATATGMNNATCACTATCCGGA; SEQ ID NO. 10 40F: TAGTGATATTCATATTCTGNNKGTTGAGGGTTTTC; SEQ ID NO. 11 40R: GAAAACCCTCAACMNNCAGAATATGAATATCACTA; SEQ ID NO. 12 42F: TATTCATATTCTGATTGTTNNKGGTTTTCTGCTGT; SEQ ID NO. 13 42R: ACAGCAGAAAACCMNNAACAATCAGAATATGAATA; SEQ ID NO. 14 43F: TCATATTCTGATTGTTGAGNNKTTTCTGCTGTATA; SEQ ID NO. 15 43R: TATACAGCAGAAAMNNCTCAACAATCAGAATATGA; SEQ ID NO. 16 44F: TATTCTGATTGTTGAGGGTNNKCTGCTGTATAATT; SEQ ID NO. 17 44R: AATTATACAGCAGMNNACCCTCAACAATCAGAATA; SEQ ID NO. 18 46F: GATTGTTGAGGGTTTTCTGNNKTATAATTATAAAC; SEQ ID NO. 19 46R: GTTTATAATTATAMNNCAGAAAACCCTCAACAATC; SEQ ID NO. 20 47F: TGTTGAGGGTTTTCTGCTGNNKAATTATAAACCGC; SEQ ID NO. 21 47R: GCGGTTTATAATTMNNCAGCAGAAAACCCTCAACA; SEQ ID NO. 22 55F: TAAACCGCTGATTGATNNKTACAATAAATGTTTTT; SEQ ID NO. 23 55R: AAAAACATTTATTGTAMNNATCAATCAGCGGTTTA; SEQ ID NO. 24 57F: GCTGATTGATGTGTACNNKAAATGTTTTTACGTGA; SEQ ID NO. 25 57R: TCACGTAAAAACATTTMNNGTACACATCAATCAGC; SEQ ID NO. 26 59F: TGATGTGTACAATAAANNKTTTTACGTGACCATTC; SEQ ID NO. 27 59R: GAATGGTCACGTAAAAMNNTTTATTGTACACATCA; SEQ ID NO. 28 60F: TGTGTACAATAAATGTNNKTACGTGACCATTCCGT; SEQ ID NO. 29 60R: ACGGAATGGTCACGTAMNNACATTTATTGTACACA; SEQ ID NO. 30 61F: GTACAATAAATGTTTTNNKGTGACCATTCCGTATG; SEQ ID NO. 31 61R: CATACGGAATGGTCACMNNAAAACATTTATTGTAC; SEQ ID NO. 32 62F: CAATAAATGTTTTTACNNKACCATTCCGTATGAAG; SEQ ID NO. 33 62R: CTTCATACGGAATGGTMNNGTAAAAACATTTATTG; SEQ ID NO. 34 112F: CTGGATATTCAGTATNNKGATGGTATGAGCAGTAA; SEQ ID NO. 35 112R: TTACTGCTCATACCATC MNNATACTGAATATCCAG; SEQ ID NO. 36 125F: TGAACTGTATAATCAGNNKTATGAAGATATTCAGA; SEQ ID NO. 37 125R: TCTGAATATCTTCATAMNNCTGATTATACAGTTCA; SEQ ID NO. 38 129F: CAGCTGTATGAAGATNNKCAGAACAGCCTGCTGAA; SEQ ID NO. 39 129R: TTCAGCAGGCTGTTCTGMNNATCTTCATACAGCTG; SEQ ID NO. 40 133F: GATATTCAGAACAGCNNKCTGAATATTCTGGCGGA; SEQ ID NO. 41 133R: TCCGCCAGAATATTCAGMNNGCTGTTCTGAATATC; SEQ ID NO. 42 MSSFDYLKTAIKQQGCTLQQVADASGMTKGYLSQLLNAKIKSPSAQKLEALHRFLGLEFPRQKKTIGVVFGKFYPLHTGHIYLIQRACSQVDELHIIMGFDDTRDRALFEDSAMSQQPTVPDRLRWLLQTFKYQKNIRIHAFNEEGMEPYPHGWDVWSNGIKKFMAEKGIQPDLIYTSEEADAPQYMEHLGIETVLVDPKRTFMSISGAQIRENPFRYWEYIPTEVKPFFVRTVAILGGESSGKSTLVNKLANIFNTTSAWEYGRDYVFSHLGGDEIALQYSDYDKIALGHAQYIDFAVKYANKVAFIDTDFVTTQAFCKKYEGREHPFVQALIDEYRFDLVILLENNTPWVADGLRSLGSSVDRKEFQNLLVEMLEENNIEFVRVEEEDYDSRFLRCVELVREMMGEQRQLEHHHHHH*; SEQ ID NO. 43 W11 F-F: GAATACCGTTAAAGGCTTCATGGAAAATCCGGTTA; SEQ ID NO. 44 W11 F-R: TAACCGGATTTTCCATGAAGCCTTTAACGGTATTC; SEQ ID NO. 45 M12R-F: TACCGTTAAAGGCTGGAGGGAAAATCCGGTTAA; SEQ ID NO. 46 W11 F+M12R-F: TACCGTTAAAGGCTTCAGGGAAAATCCGGTTAA; SEQ ID NO. 47 M12R-R: TTAACCGGATTTTCCCTCCAGCCTTTAACGGTA; SEQ ID NO. 48 W11 F+M12R-R: TTAACCGGATTTTCCCTGAAGCCTTTAACGGTA; SEQ ID NO. 49 H37W-F: CGATCCGGATAGTGATATTTGGATTCTGATTGTTG; SEQ ID NO. 50 H37W-R: CAACAATCAGAATCCAAATATCACTATCCGGATCG; SEQ ID NO. 51 H37+I40-F: CCGGATAGTGATATTTGGATTCTGSTTGTTGAGG; SEQ ID NO. 52 H37+I40-R: CCTCAACAASCAGAATCCAAATATCACTATCCGG; SEQ ID NO. 53 I40L / V-F: TAGTGATATTCATATTCTGSTTGTTGAGGGTTTTC; SEQ ID NO. 54 I40L / V-R: GAAAACCCTCAACAASCAGAATATGAATATCACTA; SEQ ID NO. 55 G43A / D-F: TCATATTCTGATTGTTGAGGMTTTTCTGCTGTATA; SEQ ID NO. 56 G43A / D-R: TATACAGCAGAAAAKCCTCAACAATCAGAATATGA; SEQ ID NO. 57 I40+G43-F: ATTCTGSTTGTTGAGGMTTTTCTGCTGTAT; SEQ ID NO. 58 I40+G43-R: ATACAGCAGAAAAKCCTCAACAASCAGAAT; SEQ ID NO. 59 L46D / E-F: GATTGTTGAGGGTTTTCTGGASTATAATTATAAAC; SEQ ID NO. 60 L46D / E-R: GTTTATAATTATASTCCAGAAAACCCTCAACAATC; SEQ ID NO. 61 G43+L46-F: TGTTGAGGMTTTTCTGGASTATAATTATAAACCG; SEQ ID NO. 62 G43+L46-R: CGGTTTATAATTATASTCCAGAAAAKCCTCAACA; SEQ ID NO. 63 V55D / E-F: TAAACCGCTGATTGATGAKTACAATAAATG; SEQ ID NO. 64 V55D / E-R: CATTTATTGTAMTCATCAATCAGCGGTTTA; SEQ ID NO. 65 F60W+Y61S-F: TACAATAAATGTTGGTCCGTGACCATTCCGTATG; SEQ ID NO. 66 Y61S-F: TACAATAAATGTTTTTCCGTGACCATTCCGTATG; SEQ ID NO. 67 F60W+Y61S-R: CATACGGAATGGTCACGGAAAAACATTTATTGTA; SEQ ID NO. 68 Y61S-R: CATACGGAATGGTCACGGAAAAACATTTATTGTA; SEQ ID NO. 69 F60W-F: TACAATAAATGTTGGTACGTGACCATTCCGT; SEQ ID NO. 70 F60W-R: ACGGAATGGTCACGTACCAACATTTATTGTA; SEQ ID NO. 71 L112I / K-F: CTGGATATTCAGTATAWSGATGGTATGAGCAGTAA; SEQ ID NO. 72 L112I / K-R: TTACTGCTCATACCATCSWTATACTGAATATCCAG; SEQ ID NO. 73 L125E-F: TGAACTGTATAATCAGGAGTATGAAGATA; SEQ ID NO. 74 L125E-R: TATCTTCATACTCCTGATTATACAGTTCA; SEQ ID NO. 75 L133T-F: TTCAGAACAGCACGCTGAATATTCTGGCGGA; SEQ ID NO. 76 L133T-R: TCCGCCAGAATATTCAGCGTGCTGTTCTGAA.

[0050] SEQ ID NO. 77 MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEDFLLYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 78 MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEGFLEYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 79 MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILVVEDFLLYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 80 MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEGFLEYNYKPLIDVYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 81 MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILVVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 82 MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEGFLEYNYKPLIDVYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYKDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 83 MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEGFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQLYEDIQNSTLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 84 MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIWILIVEDFLEYNYKPLIDVYNKCWYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYKDGMSSKDELYNQEYEDIQNSTLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 85 MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*; SEQ ID NO. 86 MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIWILLVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*.

[0051] The above embodiments are the preferred implementation of the present application, in addition to which the present application can be implemented in other manners, and any obvious substitutions within the concept of the present application are within the protection scope of the present application.

Claims

1. A mutant of nicotinamide adenine dinucleotide synthase, characterized in that, The mutant is obtained by mutating a gene that edits the amino acid sequence of drNadR as shown in SEQ ID NO.1, and contains any one of the mutation sites W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, and L133T.

2. The mutant of nicotinamide adenine dinucleotide synthase according to claim 1, characterized in that, The amino acid sequence of the nicotinamide adenine dinucleotide synthase mutant is shown in any one of SEQ ID NO.77 to SEQ ID NO.

86.

3. A nucleic acid molecule, characterized in that, The nucleotide sequence of the nucleic acid molecule refers to the nucleotide sequence encoding the nicotinamide adenine dinucleotide synthase mutant as described in any one of claims 1 to 2.

4. A carrier, characterized in that, The vector can express the nicotinamide adenine dinucleotide synthase mutant as described in any one of claims 1 to 2 or contain the nucleic acid molecule as described in claim 3.

5. A recombinant cell, characterized in that, The recombinant cells comprise the vector as described in claim 4.

6. A method for preparing a mutant of nicotinamide adenine dinucleotide synthase, characterized in that, Nicotinamide adenine dinucleotide synthase mutants were obtained by culturing the recombinant cells as described in claim 5.

7. A method for preparing nicotinamide adenine dinucleotide, characterized in that, Nicotinamide adenine dinucleotide synthase mutant as described in any one of claims 1 to 2 is used to catalyze the production of nicotinamide adenine dinucleotide with nicotinamide ribose and adenosine triphosphate.

8. The application of a nicotinamide adenine dinucleotide synthase, characterized in that, Application of the nicotinamide adenine dinucleotide synthase mutant as described in any one of claims 1 to 2 in the preparation of nicotinamide adenine dinucleotide.

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