A mutant of nicotinamide adenine dinucleotide synthetase and use thereof

Abstract

The present application relates to the technical field of biological enzyme engineering, and particularly relates to a nicotinamide adenine dinucleotide synthetase mutant and application thereof. The nicotinamide adenine dinucleotide synthetase mutant is obtained by mutating W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, L133T and other sites of NadR as a parent, so that the mutant can efficiently catalyze low-cost substrates NR and ATP to generate NAD. In particular, by using the above-mentioned multi-mutation point mutation, the mutant has better substrate binding capacity and catalytic efficiency, 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 significantly reduces the production cost of NAD by using the low-priced NR to replace the expensive NMN as the substrate, and provides a more economically beneficial technical scheme for the 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 condition, energy saving and environmental protection, and no organic solvent residue. 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:

[0009] 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

[0010] The present application will be further described below in conjunction with the drawings and examples.

[0011] Figure 1 is the liquid chromatogram of the reaction result of Example 8;

[0012] Figure 2 is the liquid chromatogram of the standard;

[0013] Figure 3 is Figure 1 and Figure 2 the comparison chart of DETAILED DESCRIPTION

[0014] As used herein, the term "and / or" includes the term "and" and the term "or". The terms used herein are used only to describe specific embodiments, and are not intended to limit the present application. As used herein, the singular forms "a", "an" and "the" are also 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.

[0015] 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 is 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 not in an idealized or overly formal sense, unless expressly so defined herein.

[0016] 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," and the like are to be construed to be open-ended and non-limiting. Furthermore, the terms of expression used herein are used as descriptions and not limitations, and it is not intended that any equivalent characteristics be excluded by the use of these terms of expression, but rather that part of the characteristics are described, but various modifications are possible within the scope of the present application according to the right. Thus, although the present application has been specifically disclosed by preferred embodiments and optional features, modifications to the disclosed embodiments to embody the application in its various aspects can be made by those skilled in the art, and such modifications and variations will be considered to be within the scope of the present application.

[0017] 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, and there is no 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, as long as they can play the expected role, and there is no particular limitation. 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.

[0018] The present application first proposes a mutant of nicotinamide adenine dinucleotide synthetase. The mutant is obtained by site-directed mutagenesis of the amino acid sequence of zebrafish NadR, introducing mutations at specific sites (such as W11F, M12R, etc.), and constructing and expressing by genetic engineering means. The mutant has higher catalytic activity and can improve the synthesis efficiency of NMN.

[0019] Example 1

[0020] The zebrafish NadR amino acid sequence (drNadR) is shown in SEQ ID NO. 1.

[0021] The preparation method of the mutant of nicotinamide adenine dinucleotide synthetase comprises:

[0022] S1. Constructing an original recombinant plasmid:

[0023] (1) obtaining the amino acid sequence corresponding to the NadR gene from Danio rerio;

[0024] (2) codon optimization and synthesis of the gene sequence of the amino acid sequence;

[0025] (3) constructing the gene sequence into pET22b(+) vector to obtain recombinant plasmid pET22b(+)-drNadR;

[0026] S2. Determining the mutation site:

[0027] The substrate, product and three-dimensional structure of the drNadR amino acid sequence are analyzed by a molecular docking method to determine the mutation site in the active pocket;

[0028] S3. Perform site-directed mutagenesis:

[0029] The recombinant plasmid is subjected to site-directed mutagenesis, and after DpnI enzyme digestion, the mutated recombinant plasmid is obtained;

[0030] S4. Obtain the mutant:

[0031] The mutated recombinant plasmid is transformed into an expression strain, cultured and induced to express to obtain the mutant.

[0032] In step S1, the amino acid sequence corresponding to the NadR gene from Danio rerio (Genebank: NP_001004618) is screened out in the NCBI library, codon optimization and gene sequence synthesis are performed in Universal Biological (Anhui) Co., Ltd., and the recombinant plasmid pET22b (+) -drNadR is obtained. 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.

[0033] In step S2, the three-dimensional structures of the substrate NR, product NAD and 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 after screening, the first round of saturation mutagenesis is determined, and 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 mutagenesis PCR; after the PCR reaction, whole DMT Enzyme (DpnI) is used for digestion, and after digestion, it is directly transformed into the DH5a strain, single colonies are selected and sequenced.

[0034] 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, L133T. When the site-directed mutation is a single-point mutation, the primers used are as shown in SEQ ID NO.2-41.

[0035] Example 2

[0036] Using a method similar to that in Example 1, a comparative plasmid was constructed:

[0037] 1. The NadR gene was amplified from the Escherichia coli genome using primer PCR, and restriction enzyme sites NdeI and XhoI were added to both ends, respectively;

[0038] The primer sequences are as follows:

[0039] ecNadR-F: 5'-GGAATTCCATATGTCGTCATTTGATTAC-3' and ecNadR-R: 5'-CCGCTCGAGTTGTCTCTGCTCCCCCCATC-3';

[0040] 2. The recombinant plasmid pET22b(+)-ecNadR was obtained by ligating the Escherichia coli NadR gene with plasmid pET22b(+) via enzyme digestion.

[0041] 3. The obtained recombinant plasmid pET22b(+)-ecNadR was transformed into DH5α competent cells. Single clones were selected for colony PCR screening. Positive single clones were selected for preservation and plasmid extraction. The amino acid sequence was confirmed to be correct by sequencing.

[0042] The positive recombinant plasmid pET22b(+)-ecNadR was transformed into the expression host bacterium BL21(DE3) to obtain the expression strain ecNadR, the amino acid sequence of which is shown in SEQ ID NO.42.

[0043] Example 3

[0044] The enzyme activities of ecNadR, drNadR, and their mutants were determined using the above method.

[0045] It should be noted that an enzyme activity unit (U) is defined as the amount of enzyme required to convert 1 micromolar nicotinamide ribose into nicotinamide adenine dinucleotide within 1 minute at 37°C.

[0046] Test method: Dilute the enzyme solution to 1 mg / mL, and add 100 μL of the enzyme solution to 100 μL of the reaction solution (final concentration is pH 6.0, 100 mM potassium dihydrogen phosphate buffer, 60 mM magnesium chloride, 120 mM nicotinamide ribose and 120 mM adenosine triphosphate), and react at 37℃ for 5 min.

[0047] After the reaction was completed, the molar concentration of nicotinamide adenine dinucleotide (NAD) in the reaction solution was determined by high performance liquid chromatography (HPLC). The results are shown in Table 1 below.

[0048] Table 1

[0049]

[0050] As shown in Table 1, the drNadR mutants modified by single-point mutation all exhibited significantly enhanced catalytic activity. Specifically, the NAD production (7.01–8.62 mM) and enzyme activity (2.80–3.45 U / mg) of all mutants were significantly higher than those of the original ecNadR (0.01 mM, 0.01 U / mg) and drNadR (5.11 mM, 2.04 U / mg).

[0051] Example 4

[0052] The site-directed mutagenesis was a multi-point mutagenesis, and the primers used are shown in SEQ ID NO.43-76. Using the drNadR plasmid as a template, primers were designed to perform combined mutagenesis of W11F, M12R, H37W, I40V, I40L, G43A, G43D, L46D, L46E, V55D, V55E, F60W, Y61S, L112I, L112K, L125E, and L133T. 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.

[0053] Table 2

[0054]

[0055] Example 5

[0056] Enzyme activity of mutant drNadR-M12R / H37W / I40L / L46E / V55E / L112I / L125E: Dissolve 34.92g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, and 1.02g potassium dihydrogen phosphate in 646ml of water, and adjust the pH to 6.0. Then add 354ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0057] The result was the production of 41.3 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 23.33 U / mg.

[0058] Example 6

[0059] Enzyme activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: Dissolve 34.92g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, and 1.02g potassium dihydrogen phosphate in 646ml of water, and adjust the pH to 6.0. Then add 354ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0060] The result was the production of 52.6 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 29.72 U / mg.

[0061] Example 7

[0062] Enzyme activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: Dissolve 34.92g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, and 1.02g potassium dihydrogen phosphate in 646ml of water, and adjust the pH to 5.5. Then add 354ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0063] The result was the production of 62.4 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 35.25 U / mg.

[0064] Example 8

[0065] Enzyme activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: Dissolve 34.92g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, 1.02g potassium dihydrogen phosphate, and 3.2g ammonium chloride in 646ml of water, adjusting the pH to 5.5. Add 354ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0066] The result was the production of 72.3 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 40.85 U / mg.

[0067] Example 9

[0068] Enzyme activity of mutant drNadR-M12R / H37W / I40L / G43D / V55E / L112I / L125E: Dissolve 58.2g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, 1.02g potassium dihydrogen phosphate, and 3.2g ammonium chloride in 820ml of water, adjusting the pH to 5.5. Add 180ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0069] The result was the generation of 99.26 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 110.29 U / mg.

[0070] Example 10

[0071] Enzyme activity of control strain ecNadR:

[0072] Dissolve 34.92g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, 1.02g potassium dihydrogen phosphate, and 3.2g ammonium chloride in 646ml of water and adjust the pH to 5.5. Then add 354ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0073] The result was the production of 0.32 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 0.18 U / mg.

[0074] Example 11

[0075] Enzyme activity of control strain ecNadR:

[0076] Dissolve 58.2g nicotinamide ribose, 66.14g adenosine triphosphate, 12.2g magnesium chloride, 9.7g dipotassium hydrogen phosphate, 1.02g potassium dihydrogen phosphate, and 3.2g ammonium chloride in 820ml of water and adjust the pH to 5.5. Then add 180ml of enzyme solution, place in a 37℃ water bath, and mechanically stir at 200rpm for 5 minutes.

[0077] The result was the production of 0.40 mM nicotinamide adenine dinucleotide, and the enzyme activity of the mutant protein was 0.44 U / mg.

[0078] Examples 5 and 6 compare the enzyme activities of two strains with the highest enzyme activity after mutation under the same reaction conditions.

[0079] Examples 6 and 7: The pH value in the reaction system was adjusted to decrease from 6.0 to 5.5, and the enzyme activity increased from 29.72 U / mg to 35.25 U / mg;

[0080] Examples 7 and 8: Adding ammonium chloride to the enzyme catalytic system increased the enzyme activity from 35.25 U / mg to 40.85 U / mg;

[0081] Examples 8 and 9: Increasing the concentration of the substrate nicotinamide ribose (NR) while decreasing the enzyme dosage resulted in an enzyme activity of 110.29 U / mg;

[0082] Examples 8 and 10: Comparing the enzyme activities of drNadR mutant and ecNadR under the same reaction conditions, the enzyme activity of drNadR mutant is much greater than that of ecNadR.

[0083] Examples 9 and 11: Comparison of enzyme activities of drNadR mutant and ecNadR under the same reaction conditions. Again, the enzyme activity of drNadR mutant is much greater than that of ecNadR.

[0084] As can be seen from Examples 3-11, the multi-site combined mutation strains significantly improved the catalytic performance of drNadR, which was more significant than that of single-point mutations.

[0085] This includes:

[0086] M12R / H37W / I40L / G43D / V55E / L112I / L125E,

[0087] The two seven-site mutation combinations, M12R / H37W / I40L / L46E / V55E / L112I / L125E, showed the best performance, with NAD production reaching 31.22 mM and 30.48 mM, respectively, and enzyme activities reaching 12.49 U / mg and 12.19 U / mg, respectively.

[0088] The above values ​​represent a significant improvement over both the original enzyme and the single-point mutant. This indicates that the combined mutations at these key sites have a synergistic effect, successfully achieving a significant optimization of enzyme activity.

[0089] SEQ ID NO.1

[0090] MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIHILIVEGFLLYNYKPLIDVYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQLYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0091] SEQ ID NO.2

[0092] 11F: GAATACCGTTAAAGGCNNKATGGAAAATCCGGTTA;

[0093] SEQ ID NO.3

[0094] 11R: TAACCGGATTTTCCATMNNGCCTTTAACGGTATTC;

[0095] SEQ ID NO.4

[0096] 12F: TACCGTTAAAGGCTGGNNKGAAAATCCGGTTAA;

[0097] SEQ ID NO.5

[0098] 12R: TTAACCGGATTTTCMNNCCAGCCTTTAACGGTA;

[0099] SEQ ID NO.6

[0100] 37F: CGATCCGGATAGTGATATTNNKATTCTGATTGTTG;

[0101] SEQ ID NO.7

[0102] 37R: CAACAATCAGAATMNNAATATCACTATCCGGATCG;

[0103] SEQ ID NO.8

[0104] 38F: TCCGGATAGTGATNNKCATATTCTGATTGTTGAGG;

[0105] SEQ ID NO.9

[0106] 38R:CCTCAACAATCAGAATATGMNNATCACTATCCGGA;

[0107] SEQ ID NO.10

[0108] 40F:TAGTGATATTCATATTCTGNNKGTTGAGGGTTTTC;

[0109] SEQ ID NO.11

[0110] 40R:GAAAACCCTCAACMNNCAGAATATGAATATCACTA;

[0111] SEQ ID NO.12

[0112] 42F:TATTCATATTCTGATTGTTNNKGGTTTTCTGCTGT;

[0113] SEQ ID NO.13

[0114] 42R:ACAGCAGAAAACCMNNAACAATCAATCAATGAATA;

[0115] SEQ ID NO.14

[0116] 43F:TCATATTCTGATTGTTGAGNNCTTTCTGCTGTATA;

[0117] SEQ ID NO.15

[0118] 43R:TATACAGCAGAAAMNNCTCAACAATCAATGAATGA;

[0119] SEQ ID NO.16

[0120] 44F:TATTCTGATTGTTGAGGGTNNKCTGCTGTATAATT;

[0121] SEQ ID NO.17

[0122] 44R:AATTATACAGCAGMNNACCCTCAACAATCAGAATA;

[0123] SEQ ID NO.18

[0124] 46F:GATTGTTGAGGTTTTCTGNNCTATAATTAAAC;

[0125] SEQ ID NO.19

[0126] 46R:GTTTATATTATAMNNCAGAAAACCCTCAACAATC;

[0127] SEQ ID NO.20

[0128] 47F:TGTTGAGGGTTTTCTGCTGNNKAATTAAACCGC;

[0129] SEQ ID NO.21

[0130] 47R:GCGGTTTATAATTMNNCAGCAGAAAACCCTCAACA;

[0131] SEQ ID NO.22

[0132] 55F:TAAACCGCTGATTGATNNKTACAATAAATGTTTTT;

[0133] SEQ ID NO.23

[0134] 55R:AAAAACATTTATTGTAMNNATCAATCAGCGGTTTA;

[0135] SEQ ID NO.24

[0136] 57F:GCTGATTGATGTGTACNNKAAATGTTTTTACGTGA;

[0137] SEQ ID NO.25

[0138] 57R:TCACGTAAAAACATTTMNNGTACACATCAATCAGC;

[0139] SEQ ID NO.26

[0140] 59F:TGATGTGTACAATAAANNKTTTTACGTGACCATTC;

[0141] SEQ ID NO.27

[0142] 59R:GAATGGTCACGTAAAAMNNTTTATTGTACACATCA;

[0143] SEQ ID NO.28

[0144] 60F:TGTGTACAATAAATGTNNKTACGTGACCATTCCGT;

[0145] SEQ ID NO.29

[0146] 60R:ACGGAATGGTCACGTAMNNACATTTATTGTACACA;

[0147] SEQ ID NO.30

[0148] 61F:GTACAATAAATGTTTTNNKGTGACCATTCCGTATG;

[0149] SEQ ID NO.31

[0150] 61R:CATACGGAATGGTCACMNNAAAACATTTATTGTAC;

[0151] SEQ ID NO.32

[0152] 62F:CAATAAATGTTTTTACNNKACCATTCCGTATGAAG;

[0153] SEQ ID NO.33

[0154] 62R:CTTCATACGGAATGGTMNNGTAAAAACATTTATTG;

[0155] SEQ ID NO.34

[0156] 112F:CTGGATATTCAGTATNNKGATGGTATGAGCAGTAA;

[0157] SEQ ID NO.35

[0158] 112R:TTACTGCTCATACCATCMNNATACTGAATATCCAG;

[0159] SEQ ID NO.36

[0160] 125F:TGAACTGTATAATCAGNNKTATGAAGATATTCAGA;

[0161] SEQ ID NO.37

[0162] 125R:TCTGAATATCTTCATAMNNCTGATTATACAGTTCA;

[0163] SEQ ID NO.38

[0164] 129F:CAGCTGTATGAAGATNNKCAGAACAGCCTGCTGAA;

[0165] SEQ ID NO.39

[0166] 129R:TTCAGCAGGCTGTTCTGMNNATCTTCATACAGCTG;

[0167] SEQ ID NO.40

[0168] 133F:GATATTCAGAACAGCNNKCTGAATATTCTGGCGGA;

[0169] SEQ ID NO.41

[0170] 133R:TCCGCCAGAATATTCAGMNNGCTGTTCTGAATATC;

[0171] SEQ ID NO.42

[0172] MSSFDYLKTAIKQQGCTLQQVADASGMTKGYLSQLLNAKIKSPSAQKLEALHRFLGLEFPRQKKTIGVVFGKFYPLHTGHIYLIQRACSQVDELHIIMGFDDTRDRALFEDSAMSQQPTVPDRLRWLLQTFKYQKNIRIHAFNEEGMEPYPHGWDVWSNGIKKFMAEKGIQPDLIYTSEEADAPQYMEHLGIETVLVDPKRTFMSISGAQIRENPFRYWEYIPTEVKPFFVRTVAILGGESSGKSTLVNKLANIFNTTSAWEYGRDYVFSHLGGDEIALQYSDYDKIALGHAQYIDFAVKYANKVAFIDTDFVTTQAFCKKYEGREHPFVQALIDEYRFDLVILLENNTPWVADGLRSLGSSVDRKEFQNLLVEMLEENNIEFVRVEEEDYDSRFLRCVELVREMMGEQRQLEHHHHHH*;

[0173] SEQ ID NO.43

[0174] W11F-F:GAATACCGTTAAAGGCTTCATGGAAAATCCGGTTA;

[0175] SEQ ID NO.44

[0176] W11F-R:TAACCGGATTTTCCATGAAGCCTTTAACGGTATTC;

[0177] SEQ ID NO.45

[0178] M12R-F:TACCGTTAAAGGCTGGAGGGAAAATCCGGTTAA;

[0179] SEQ ID NO.46

[0180] W11F+M12R-F:TACCGTTAAAGGCTTCAGGGAAAATCCGGTTAA;

[0181] SEQ ID NO.47

[0182] M12R-R:TTAACCGGATTTTCCCTCCAGCCTTTAACGGTA;

[0183] SEQ ID NO.48

[0184] W11F+M12R-R:TTAACCGGATTTTCCCTGAAGCCTTTAACGGTA;

[0185] SEQ ID NO.49

[0186] H37W-F:CGATCCGGATAGTGATATTTGGATTCTGATTGTTG;

[0187] SEQ ID NO.50

[0188] H37W-R:CAACAATCAGAATCCAAATATCACTATCCGGATCG;

[0189] SEQ ID NO.51

[0190] H37+I40-F:CCGGATAGTGATATTTGGATTCTGSTTGTTGAGG;

[0191] SEQ ID NO.52

[0192] H37+I40-R:CCTCAACAASCAGAATCCAAATATCACTATCCGG;

[0193] SEQ ID NO.53

[0194] I40L / V-F:TAGTGATATTCATATTCTGSTTGTTGAGGGTTTTC;

[0195] SEQ ID NO.54

[0196] I40L / V-R:GAAAACCCTCAACAASCAGAATATGAATATCACTA;

[0197] SEQ ID NO.55

[0198] G43A / DF:TCATATTCTGATTGTTGAGGMTTTTCTGCTGTATA.

[0199] SEQ ID NO.56

[0200] G43A / DR:TATACAGCAGAAAAKCCTCAACAATCAGAATATGA.

[0201] SEQ ID NO.57

[0202] I40+G43-F:ATTCTGSTTGTTGAGGMTTTTCTGCTGTAT;

[0203] SEQ ID NO.58

[0204] I40+G43-R:ATACAGCAGAAAAKCCTCAACAASCAGAAT;

[0205] SEQ ID NO.59

[0206] L46D / EF:GATTGTTGAGGGTTTTCTGGASTATAATTATAAAC;

[0207] SEQ ID NO.60

[0208] L46D / ER:GTTTATAATTATASTCCAGAAAACCCTCAACAATC;

[0209] SEQ ID NO.61

[0210] G43+L46-F:TGTTGAGGMTTTTCTGGASTATAATTTATAAACCG;

[0211] SEQ ID NO.62

[0212] G43+L46-R:CGGTTTATAATTTATASTCCAGAAAAKCCTCAACA;

[0213] SEQ ID NO.63

[0214] V55D / EF:TAAACCGCTGATTGATGAKTACAATAAATG.

[0215] SEQ ID NO.64

[0216] V55D / E-R:CATTTATTGTAMTCATCAATCAGCGGTTTA;

[0217] SEQ ID NO.65

[0218] F60W+Y61S-F:TACAATAAATGTTGGTCCGTGACCATTCCGTATG;

[0219] SEQ ID NO.66

[0220] Y61S-F:TACAATAAATGTTTTTCCGTGACCATTCCGTATG;

[0221] SEQ ID NO.67

[0222] F60W+Y61S-R:CATACGGAATGGTCACGGACCAACATTTATTGTA;

[0223] SEQ ID NO.68

[0224] Y61S-R:CATACGGAATGGTCACGGAAAAACATTTATTGTA;

[0225] SEQ ID NO.69

[0226] F60W-F:TACAATAAATGTTGGTACGTGACCATTCCGT;

[0227] SEQ ID NO.70

[0228] F60W-R:ACGGAATGGTCACGTACCAACATTTATTGTA;

[0229] SEQ ID NO.71

[0230] L112I / K-F:CTGGATATTCAGTATAWSGATGGTATGAGCAGTAA;

[0231] SEQ ID NO.72

[0232] L112I / K-R:TTACTGCTCATACCATCSWTATACTGAATATCCAG;

[0233] SEQ ID NO.73

[0234] L125E-F:TGAACTGTATAATCAGGAGTATGAAGATA;

[0235] SEQ ID NO.74

[0236] L125E-R:TATCTTCATACTCCTGATTATACAGTTCA;

[0237] SEQ ID NO.75

[0238] L133T-F:TTCAGAACAGCACGCTGAATATTCTGGCGGA;

[0239] SEQ ID NO.76

[0240] L133T-R:TCCGCCAGAATATTCAGCGTGCTGTTCTGAA。

[0241] SEQ ID NO.77

[0242] MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEDFLLYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0243] SEQ ID NO.78

[0244] MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEGFLEYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0245] SEQ ID NO.79

[0246] MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILVVEDFLLYNYKPLIDEYNKCFYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0247] SEQ ID NO.80

[0248] MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILLVEGFLEYNYKPLIDVYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0249] SEQ ID NO.81

[0250] MDAMVNTVKGWRENPVKFARSHGVSVSTTSDPDSDIWILVVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0251] SEQ ID NO.82

[0252] MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEGFLEYNYKPLIDVYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYKDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0253] SEQ ID NO.83

[0254] MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEGFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYIDGMSSKDELYNQLYEDIQNSTLNILADPNSSSVDKLAAALEHHHHHH*;

[0255] SEQ ID NO.84

[0256] MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIWILIVEDFLEYNYKPLIDVYNKCWYVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYKDGMSSKDELYNQEYEDIQNSTLNILADPNSSSVDKLAAALEHHHHHH*;

[0257] SEQ ID NO.85

[0258] MDAMVNTVKGFMENPVKFARSHGVSVSTTSDPDSDIWILIVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*;

[0259] SEQ ID NO.86

[0260] MDAMVNTVKGWMENPVKFARSHGVSVSTTSDPDSDIWILLVEDFLLYNYKPLIDEYNKCFSVTIPYEECKRRRSTRTYTVPDPPGLFDGHVWPMYLKHRTEMENSSLDIQYLDGMSSKDELYNQEYEDIQNSLLNILADPNSSSVDKLAAALEHHHHHH*.

[0261] The above embodiments are preferred implementations of the present invention. In addition, the present invention can be implemented in other ways. Any obvious substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A mutant of nicotinamide adenine dinucleotide synthase, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO.

77.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule contains a nucleotide sequence encoding the mutant as described in claim 1.

3. A carrier, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the vector as described in claim 3.

5. 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 4.

6. A method for preparing nicotinamide adenine dinucleotide, characterized in that, Using the nicotinamide adenine dinucleotide synthase mutant as described in claim 1, nicotinamide ribose and adenosine triphosphate are used as substrates to catalyze the synthesis of nicotinamide adenine dinucleotide products.

7. The application of a mutant nicotinamide adenine dinucleotide synthase, characterized in that, The nicotinamide adenine dinucleotide synthase mutant as described in claim 1 is applied to the catalytic synthesis of nicotinamide adenine dinucleotide products using nicotinamide ribose and adenosine triphosphate as substrates.

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