NMNAT enzyme mutant and application thereof in NAD < + > biosynthesis

Abstract

The invention relates to the technical field of biology, in particular to an NMNAT enzyme mutant and application of the NMNAT enzyme mutant in NAD < + > synthesis. According to the invention, an active pocket of NMNAT enzyme is modified to screen out some single-point mutation or multi-point combined mutation nicotinamide mononucleotide adenosine transferase mutants, and the mutants can convert high-concentration NMN to synthesize NAD (Nicotinamide Adenosine Dinucleotide). The invention aims to solve the problem that the substrate concentration of the current NMN adenylation reaction is generally very low, realizes efficient NMN adenylation synthesis of NAD < + >, and can be applied to actual production and application of NAD < + >.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to an NMNAT enzyme mutant and its application in NAD+ synthesis. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD or NAD+), as an important coenzyme in cells, can transfer electrons, connect the citric acid cycle and the electron transport chain, and participate in the catalytic reactions of various oxidoreductases to maintain the intracellular redox level and balance the activities of various enzymes; in the regulation of cell signal transduction, NAD acts as a signal molecule, binds to some specific enzymes, regulates the activities and functions of the enzymes, and thus affects key processes such as cell growth, differentiation, and apoptosis.

[0003] In addition, the demand for NAD in the industrial field is huge. Biocatalysis technology mimics the reactions completed by enzymes in living organisms, and the reaction conditions are strictly carried out according to the metabolic characteristics in living organisms. For example, the chiral reduction catalyzed by common leucine dehydrogenase, formate dehydrogenase, glucose dehydrogenase, and some ketoreductases all require the participation of NAD to complete the whole reaction. It can be seen that NAD is one of the essential coenzymes in modern biocatalytic reactions. The uses of NAD include, but are not limited to, applications in chemical catalytic reactions, production of bulk drugs, health care products industry, cosmetics industry, etc., and the market demand is very large.

[0004] Currently, the methods for industrial production of NAD generally include chemical synthesis and biosynthesis. The biological method is further divided into microbial fermentation method and enzyme-catalyzed synthesis. The chemical method uses nicotinamide as a raw material and synthesizes NAD through multiple steps. The reaction route is long, and expensive reagents and a large amount of organic solvents are used, causing environmental pollution. The microbial fermentation method has relatively mature process technology, but the raw material consumption is huge, the energy consumption is large, and the output is limited. The biocatalytic method has gradually become the mainstream due to its mild reaction conditions, energy conservation and environmental protection, and no residual organic solvents. The enzymatic synthesis route of NAD uses β-nicotinamide mononucleotide (NMN) and adenosine triphosphate (ATP) as substrates and is catalyzed by nicotinamide mononucleotide adenylyltransferase (NMNAT) to synthesize NAD.

[0005] However, the substrate concentrations of the reported NMN adenylation reactions are not very high. In the examples described in Patent CN103710321B, the NMN concentration is 5 mM, which is approximately 1.6 g / L in terms of mass concentration. Considering that the ideal substrate concentration of general chemicals during industrial production is greater than 100 g / L, if the substrate concentration catalyzed by this enzyme can be increased, then the production capacity can be significantly improved, the production cost can be saved, and the goal of replacing chemical production with biological production can be achieved. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide an NMNAT enzyme mutant and its application in the preparation of NAD+.

[0007] In the mutant of the NMNAT enzyme provided by the present invention, the NMNAT enzyme is from Methanosphaera stadtmana;

[0008] The mutation sites of the mutant of the present invention include at least one of the 62nd, 125th, and 155th positions.

[0009] The 62nd residue of the NMNAT enzyme from Methanosphaera stadtmana is alanine (Ala, A), and in the mutant of the present invention, it is mutated to threonine (Thr, T) or aspartic acid (Asp, D);

[0010] The 78th residue of the NMNAT enzyme from Methanosphaera stadtmana is leucine (Leu, L), and in the mutant of the present invention, it is mutated to threonine (Thr, T);

[0011] The 108th residue of the NMNAT enzyme from Methanosphaera stadtmana is valine (Val, V), and in the mutant of the present invention, it is mutated to alanine (Ala, A) or leucine (Leu, L);

[0012] The 125th residue of the NMNAT enzyme from Methanosphaera stadtmana is phenylalanine (Phe, F), and in the mutant of the present invention, it is mutated to tyrosine (Tyr, Y);

[0013] The 155th residue of the NMNAT enzyme from Methanosphaera stadtmana is lysine (Lys, K), and in the mutant of the present invention, it is mutated to glutamic acid (Glu, E) or glycine (Gly, G).

[0014] In some embodiments, the mutant comprises at least one of the following mutation sites: A62T, A62D, L78T, V108A, V108L, F125Y, K155E or K155G.

[0015] The present invention screens the sites of wild-type NMNAT from Methanosphaera stadtmana, and site-directed mutagenesis is performed on one or more sites related to catalysis to obtain mutants capable of catalyzing the adenylation of high-concentration NMN to synthesize NAD.

[0016] In some embodiments of the present invention, if the number of mutation sites in the mutant is 2, the mutation sites included in the mutant are A62T and L78T, or A62T and V108A, or A62T and V108L, or A62T and F125Y, or A62T and K155E, or A62T and K155G, or A62D and L78T, or A62D and V108A, or A62D and V108L, or A62D and F125Y, or A62D and K155E, or A62D and K155G, or L78T and V108A, or L78T and V108L, or L78T and F125Y, or L78T and K155E, or L78T and K155G, or V108A and F125Y, or V108A and K155E, or V108A and K155G, or V108L and F125Y, or V108L and K155E, or V108L and K155G, or F125Y and K155E, or F125Y and K155G.

[0017] In some embodiments, in the mutant of the present invention, the number of mutation sites is 3, and the mutation sites included in the mutant are A62T, L78T, and V108A, or A62T, L78T, and V108L, or A62D, L78T, and V108A, or A62D, L78T, and V108L, or A62T, L78T, and F125Y, or A62D, L78T, and F125Y, or, or A62T, L78T, and K155E, or A62T, L78T, and K155G, or A62D, L78T, and K155E, or A62D, L78T, and K155G, or A62T, V108A, and F125Y, or A62T, V108L, and F125Y, or A62D, V108A, and F125Y, or A62D, V108L, and F125Y, or A62T, V108A, and K155E, or A62T, V108A, and K155G, or A62T, V108L, and K155E, or A62T, V108L, and K155G, or A62D, V108A, and K155E, or A62D, V108A, and K155G, or A62D, V108L, and K155E, or A62D, V108L, and K155G, or A62T, F125Y, and K155E, or A62T, F125Y, and K155G, or A62D, F125Y, and K155E, or A62D, F125Y, and K155G, or L78T, V108A, and F125Y, or L78T, V108L, and F125Y, or L78T, V108A, and K155E, or L78T, V108A, and K155G, or L78T, V108L, and K155E, or L78T, V108L, and K155G, or L78T, F125Y, and K155E, or L78T, F125Y, and K155G, or V108A, F125Y, and K155E, or V108A, F125Y, and K155G, or V108L, F125Y, and K155E, or V108L, F125Y, and K155G.

[0018] In some embodiments, in the mutant of the present invention, the number of mutation sites is 4, and the mutation sites included in the mutant are A62T, L78T, V108A, and F125Y, or A62T, L78T, V108L, and F125Y, or A62D, L78T, V108A, and F125Y, or A62D, L78T, V108L, and F125Y, or A62T, L78T, V108A, and K155E, or A62T, L78T, V108A, and K155G, or A62T, L78T, V108L, and K155E, or A62T, L78T, V108L, and K155G, or A62D, L78T, V108A, and K155E, or A62D, L78T, V108A, and K155G, or A62D, L78T, V108L, and K155E, or A62D, L78T, V108L, and K155G, or A62T, L78T, F125Y, and K155E, or A62T, L78T, F125Y, and K155G, or A62D, L78T, F125Y, and K155E, or A62D, L78T, F125Y, and K155G, or A62T, V108A, F125Y, and K155E, or A62T, V108A, F125Y, and K155G, or A62T, V108L, F125Y, and K155E, or A62T, V108L, F125Y, and K155G, or A62D, V108A, F125Y, and K155E, or A62D, V108A, F125Y, and K155G, or A62D, V108L, F125Y, and K155E, or A62D, V108L, F125Y, and K155G, or L78T, V108A, F125Y, and K155E, or L78T, V108A, F125Y, and K155G, or L78T, V108L, F125Y, and K155E, or L78T, V108L, F125Y, and K155G.

[0019] In some embodiments, in the mutant of the present invention, the number of mutation sites is 5, and the mutation sites included in the mutant are A62T, L78T, V108A, F125Y, and K155E, or A62T, L78T, V108A, F125Y, and K155G, or A62T, L78T, V108L, F125Y, and K155E, or A62T, L78T, V108L, F125Y, and K155G, or A62D, L78T, V108A, F125Y, and K155E, or A62D, L78T, V108A, F125Y, and K155G, or A62D, L78T, V108L, F125Y, and K155E, or A62D, L78T, V108L, F125Y, and K155G.

[0020] In some embodiments, in the mutant of the present invention, the number of mutation sites is 5, and the mutation sites included in the mutant are A62T, L78T, V108A, F125Y, and K155E, or A62T, L78T, V108A, F125Y, and K155G, or A62T, L78T, V108L, F125Y, and K155E, or A62T, L78T, V108L, F125Y, and K155G, or A62D, L78T, V108A, F125Y, and K155E, or A62D, L78T, V108A, F125Y, and K155G, or A62D, L78T, V108L, F125Y, and K155E, or A62D, L78T, V108L, F125Y, and K155G.

[0021] In some specific embodiments, the mutation sites of the mutant include A62T and F125Y; or include A62T and K155E; or include A62T, F125Y, and K155E.

[0022] In the embodiments of the present invention, the NCBI accession number of the NMNAT enzyme from Methanosphaera stadtmana is MBE6493049.1, and in the present invention, the amino acid sequence of this NMNAT enzyme is as shown in SEQ ID NO.1.

[0023] Furthermore, the present invention also provides biological materials, which include at least one of the following:

[0024] A), nucleic acid encoding the mutant as described above;

[0025] B), an expression unit including a promoter and the nucleic acid described in A);

[0026] C), a plasmid vector including the nucleic acid described in A) and / or the expression unit described in B);

[0027] D), a transformant containing the plasmid vector described in C) or having the nucleic acid described in A) and / or the expression unit described in B) integrated into its genome.

[0028] In the present invention, the nucleic acid sequence encoding the NMNAT enzyme is as shown in SEQ ID NO. 2. In the mutants:

[0029] The codon at the A62T site is ACA; the codon at the A62D site is GAC; the codon at the L78T site is ACC; the codon at the V108A site is GCA; the codon at the V108L site is CTC; the codon at the F125Y site is TAC; the codon at the K155E site is GAA; the codon at the K155G site is GGA.

[0030] In the present invention, the promoter in the expression unit is a prokaryotic promoter, a yeast promoter, an insect cell promoter or a mammalian cell promoter. For example, the promoter is: T7 promoter, lac promoter, trc promoter, GAL1 promoter, PGK1 promoter, AOX1 promoter, GAP promoter, polyhedrin promoter (polh), P10 promoter, CMV promoter, EF1α promoter or SV40 promoter.

[0031] In the present invention, the backbone vector of the plasmid vector is a pET series vector, a pBAD series vector, a pUC series vector, a pGEX series vector or a pRSF series vector. For example, it is selected from pET-11a, pET-11b, pET-11c, pET-11d, pET-14b, pET-15b, pET-16b, pET-17b, pET-19b, pET-20b(+), pET-21a(+), pET-21b(+), pET-21d(+), pET-22b(+), pET-23(+), pET-23a(+), pET-23c(+), pET-24a(+), pET-24b(+), pET-25b(+), pET-26b(+), pET-27b(+), pET-28b(+), pET-28c(+), pET-29a(+), pET-29b(+), pET-30a(+), pET-30b(+), pET-30c(+), pET-31b(+), pET-32ek / lic, pET-32a(+), pET-32b(+), pET-32c(+), pET-39b(+), pET-3a, pET-3b, pET-3c, pET-3d, pET-40b(+), pET-41a(+), pET-41b(+), pET-41ek / lic, pET-42a(+), pET-42b(+), pBAD / gIII A, pUC18, pUC19, pGEX-4T-1, pGEX-6P-1 or pRSF-Duet-1. As a feasible case, the backbone vector of the plasmid vector is pET-28a(+).

[0032] In the present invention, the host of the transformant is a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell. For example, it is at least one of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, Pichia pastoris, Spodoptera frugiperda cells, Trichoplusia ni cells, Chinese hamster ovary cells, human embryonic kidney cells or mouse myeloma cells. As a feasible case, the host is Escherichia coli. More specifically, the host is Escherichia coli E. coli BL21(DE3).

[0033] Furthermore, the present invention also provides a method for preparing the mutant as described above, which includes culturing the transformant in the biological material as described above to obtain a product containing the mutant.

[0034] Furthermore, the present invention also provides the application of the mutant as described above, the biological material or the product prepared by the preparation method in the preparation of NAD+.

[0035] Furthermore, the present invention also provides a method for preparing NAD+, which includes using β-nicotinamide mononucleotide (NMN) as a substrate and using the NMNAT enzyme mutant described in the present invention as a catalyst for an adenylation reaction. The NMNAT enzyme mutant is integrated by the genome provided by the present invention. The nucleic acid or expression unit host of the NMNAT enzyme mutant gene of the present invention is obtained by fermentation culture, or is obtained by fermentation culture of a host transfected or transformed with the plasmid vector or plasmid combination described in the present invention.

[0036] Specifically, the method for preparing NAD+ includes using NMN as a substrate and using the mutant described above as a catalyst to prepare a product containing NAD+.

[0037] In the preparation method of the present invention, the pH value of the reaction system is 7 to 7.5, wherein the concentration of NMN is 0.1 to 0.2 mol / L; the temperature of the reaction is 35 to 40 °C, and the time is 5 to 8 h.

[0038] In a specific example, the pH value of the reaction system is 7 to 7.5, wherein the concentration of NMN is 0.12 mol / L; the temperature of the reaction is 37 °C, and the time is 6 h.

[0039] In the preparation method described above, the enzyme mutant in the adenylation reaction system can be a purified NMNAT enzyme mutant or a crude enzyme solution obtained by fermentation. The present invention does not make any limitation in this regard. In a specific example, taking the crude enzyme solution as an example for verification, the concentration of the NMNAT enzyme mutant in the crude enzyme solution is 100 g / L.

[0040] More specifically, in the preparation method, the reaction system further includes ATP, magnesium ions, and pyrophosphatase. For example, the reaction system includes 0.12 mol / L - 0.16 mol / L of NMN, 0.13 mol / L - 0.18 mol / L of ATP, 0.05 mol / L of magnesium chloride hexahydrate, 100 g / L of the crude enzyme solution of the NMNAT mutant, and 100 g / L of pyrophosphatase.

[0041] The present invention modifies the active pocket of the NMNAT enzyme and screens out some single-point mutations or multi-point combination mutations of nicotinamide mononucleotide adenylyltransferase mutants, which can convert high-concentration NMN to synthesize NAD. The present invention aims to solve the problem that the substrate concentration of the current NMN adenylation reaction is generally very low, realize the efficient adenylation of NMN to synthesize NAD+, and can be applied to the actual production of NAD+. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 High-performance liquid chromatography diagram showing the NAD+ standard sample;

[0043] Figure 2 High-performance liquid chromatography (HPLC) chromatogram showing the NAN+ standard sample;

[0044] Figure 3 High-performance liquid chromatography (HPLC) chromatogram showing the reaction of NMNAT catalyzing NMN to generate NAP+;

[0045] Figure 4 High-performance liquid chromatography (HPLC) chromatogram showing the reaction of NMNAT-6 catalyzing NMN to generate NAP+;

[0046] Figure 5 High-performance liquid chromatography (HPLC) chromatogram showing the reaction of NMNAT-12 catalyzing NMN to generate NAP+. Detailed implementation manners

[0047] The present invention provides an NMNAT enzyme mutant and its application in the preparation of NAD+. Those skilled in the art can draw on the content of this article and appropriately modify the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those skilled in the art can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0048] Unless otherwise defined in the present invention, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0049] The terms "comprising", "including" and "having" can be used interchangeably, aiming to express the inclusiveness of the solution, meaning that the solution may contain other elements in addition to the listed elements. At the same time, it should be understood that when using "comprising", "including" and "having" to describe in this article, the solution of "consisting of..." is also provided.

[0050] When the term "and / or" is used in this article, it includes the meanings of "and", "or" and "any other combination of all or part of the elements linked by the term".

[0051] As used herein, the term "mutant" refers to a mutant of nicotinamide nucleotide adenylyltransferase (NNAT). NNAT is a key enzyme involved in the biosynthesis of nicotinamide adenine dinucleotide (NAD+), playing a crucial role in cellular energy metabolism, redox balance, and the maintenance of various physiological functions. In the present invention, homology modeling was performed on the NMNAT enzyme derived from Methanosphaera stadtmanae (NMNAT, NCBI accession number: MBE6493049.1, amino acid sequence shown in SEQ ID NO.1, nucleotide sequence shown in SEQ ID NO.2). Through molecular docking of the computer-simulated structure technology with the substrates NMN and ATP, it was speculated that the amino acids at positions 9, 11, 38, 39, 62, 78, 84, 91, 105, 107, 108, 111, 125, and 155 are located at the active center or the exit of the substrate pocket, and may be closely related to the catalytic activity of the enzyme. An NMNAT mutant library was constructed by means of saturation mutagenesis to screen for the optimal combination of mutant strains of NMNAT for the catalytic preparation of NAD+.

[0052] In this article, the mutation site is represented as the original amino acid - site - mutated amino acid. For example, the mutation site A62T means that in the amino acid sequence shown in SEQ ID NO:1, the 62nd amino acid is mutated from alanine to threonine. In the examples of the present invention, the " / " between mutation sites represents "and", that is, the two sites before and after " / " are mutated simultaneously; for example: A62T / F125Y means that the 62nd amino acid is mutated from alanine to threonine, and the 125th amino acid is mutated from phenylalanine to tyrosine; A62T / F125Y / K155E means that the 62nd amino acid is mutated from alanine to threonine, the 125th amino acid is mutated from phenylalanine to tyrosine; and the 155th amino acid is mutated from lysine to glutamate.

[0053] As used herein, the term "nucleic acid" refers to any compound or substance containing a nucleotide polymer. Each nucleotide consists of a base (especially a purine or pyrimidine base, such as cytosine (C), guanine (G), adenine (A), thymine (T), or uracil (U)), a sugar (such as deoxyribose or ribose), and a phosphate group. Nucleic acid molecules are usually described by their base sequences, which represent the primary structure (linear structure) of the nucleic acid and are usually represented in the 5′ to 3′ direction.

[0054] In the present invention, the term "nucleic acid molecule" encompasses deoxyribonucleic acid (DNA, including complementary DNA (cDNA) and genomic DNA), ribonucleic acid (RNA, particularly messenger RNA (mRNA)), synthetic forms of DNA or RNA, and hybrid polymers comprising two or more of these molecules. The nucleic acid molecule can be linear or circular and includes sense and antisense strands, as well as single-stranded and double-stranded forms. In addition, the nucleic acid molecules described in the present invention can contain naturally occurring nucleotides or can contain non-naturally occurring nucleotides, such as modified nucleobases with derivatized sugar or phosphate backbones. Further, the nucleic acid molecules described in the present invention include DNA (such as cDNA) or RNA (such as mRNA) suitable as vectors for direct expression of the mutants of the present invention in vitro and / or in vivo (e.g., in a host or patient). These vectors can be unmodified or chemically modified.

[0055] Expression unit: Refers to a specific combination of nucleic acid sequences capable of achieving gene expression. It generally includes a promoter that initiates gene transcription, a target gene that encodes the target product, and a terminator that ends the transcription process. This unit can drive gene transcription and translation in a suitable system to produce biomolecules such as proteins. In the present invention, in the expression unit containing the said nucleic acid, it includes a promoter and a coding nucleic acid. On this basis, the said expression unit further includes a terminator and / or an enhancer.

[0056] Vector or plasmid vector: Refers to a nucleic acid molecule capable of amplifying another nucleic acid linked thereto. This term encompasses vectors as self-replicating nucleic acid structures and vectors capable of integrating into the genome of a host cell. Some vectors can direct the expression of nucleic acids operably linked thereto. In the present invention, such vectors are referred to as "expression vectors". The said vectors can be used for storage, amplification of nucleic acids or expression units, or for the expression of truncated or fusion proteins, and the present invention does not limit this.

[0057] The nucleic acid sequences and amino acid sequences involved in the present invention are as follows:

[0058] The amino acid sequence of NMNAT derived from Methanosphaera stadtmana is:

[0059] MNEKRGLLIGRIQPLHNGHIKVIQETLKEVDEIIIGIGSAEKSHTDSNPFTGGERILMLSKALREYNVDASKYYIIPLEDISCNSLWVGHVKMLTPPFSKVYSGNSLVKQLFKENNITVIQPPLFNRTEYSGTEVRRRMLNDEDWHSLVPKSVVKVIEEIKGVQRIQNLHKKEVSELNNKKL (SEQ ID NO.1)

[0060] The nucleic acid sequence of NMNAT derived from Methanosphaera stadtmana is as follows:

[0061] CTATAATTTTTTGTTATTTAGTTCACTAACTTCCTTTTTATGTAAATTTTGTATACGTTGAACTCCTTTAATTTCTTCAATTACTTTAACAACAGATTTTGGAACTAAACTATGCCAATCTTCATCATTAAGCATTCTTCTTCTTACTTCAGTTCCAGAGTATTCTGTTCGATTAAATAGGGGTGGTTGTATAACAGTTATATTATTTTCTTTGAATAATTGTTTAACTAAGCTATTGCCTGAATATACTTTACTAAATGGTGGTGTTAGCATTTTTACATGACCTACCCATAAAGAATTACATGAAATATCTTCAAGAGGAATAATATAGTATTTAGAAGCATCAACATTATATTCACGTAATGCTTTGGATAACATTAGGATTCTTTCACCACCAGTAAATGGATTAGAATCTGTATGACTTTTTTCAGCACTTCCAATTCCTATAATGATTTCATCTACTTCTTTTAAGGTTTCTTGTATAACTTTTATATGACCATTATGTAAAGGTTGAATTCTACCTATTAATAAGCCTCTTTTTTCATTCAT(SEQ ID NO.2)

[0062] The test materials used in the present invention are all ordinary commercially available products and can be purchased on the market.

[0063] The present invention will be further described below in conjunction with embodiments:

[0064] Example 1 Screening of NMNAT Strains of Different Species

[0065] Select NMNAT protein sequences from different species on the NCBI online website and construct Escherichia coli expression strains. The specific operation steps are as follows:

[0066] Step 1 Construct strains

[0067] The nucleic acid sequences of NMNAT proteins from different species were selected through the NCBI online website and synthesized as whole genes, and a recombinant expression vector was constructed. The vector was pET-28a, and the recombinant vector was expressed in Escherichia coli BL21(DE3) to obtain a recombinant strain.

[0068] Step 2 Preparation of crude enzyme solution

[0069] Pick a single colony of the above recombinant strain and inoculate it into 5 mL of LB liquid medium containing 100 μg / mL kanamycin, and culture it with shaking at 37 °C for 12 h. Transfer it to 50 ml of LB liquid medium containing 100 μg / mL kanamycin according to an inoculation amount of 2%, and culture it with constant shaking at 37 °C for about 3 hours. When the OD 600nm is about 0.5 - 0.8, add IPTG with a final concentration of 0.4 - 0.5 mM, and induce culture at 25 °C for 16 - 24 hours. After the induction culture is completed, centrifuge the culture solution at 4 °C and 4000×g for 10 min, discard the supernatant, and collect the cells. Wash the collected cells twice with 100 mM phosphate buffer at pH 7.5, resuspend them in the phosphate buffer, and ultrasonically disrupt them 30 times at a power of 400 W, with each ultrasonic treatment lasting for 3 s and an interval of 7 s. Centrifuge this cell lysate at 12000×g and 4 °C for 10 min to remove the precipitate, and the supernatant is the crude enzyme solution.

[0070] Step 3 Determination of NMNAT enzyme activity

[0071] Using NMN as the substrate, the enzyme activity of the recombinant NMNAT strain was detected. The assay system is shown in Table 1:

[0072] Table 1 NMNAT enzyme activity assay system

[0073] Reagent Name Volume (μL) 100 mmol / L Phosphate Buffer (pH 7.5) 760 300 mmol / L NMN 100 50 mmol / L ATP 100 <![CDATA[40 mmol / L MgCl2]]> 20 Crude NMNAT Enzyme Solution 20 Final Volume 1000

[0074] All of the above substances were prepared using 100 mM phosphate buffer at pH 7.5. After the system was mixed evenly, it was placed in a constant temperature oscillator at 37 °C for reaction for 10 min, and then placed in a constant temperature oscillator at 100 °C for 5 min to terminate the reaction. Centrifuge at 12000 rpm for 5 min to remove the precipitate, and take the supernatant to measure the NAD content generated in the reaction system by high performance liquid chromatography. The determination of the enzyme activity of NMNAT protease selected from different species sources of NCBI was carried out according to Steps 1, 2, and 3. The results are shown in Table 2:

[0075] Table 2 NMNAT enzyme activities from different species sources

[0076]

[0077] Example 2 Construction of NMNAT saturation mutation library and screening of mutation sites

[0078] Select 14 active sites of NMNAT from Methanosphaera stadtmana with the highest relative enzyme activity in Example 1, namely the 9th, 11th, 38th, 39th, 62nd, 78th, 84th, 91st, 105th, 107th, 108th, 111th, 125th, and 155th positions, and perform single-site saturation mutagenesis on the above active sites.

[0079] Step 1 - Whole-plasmid PCR

[0080] Using the pET-28a(+)-NMNAT plasmid as a template, design upstream and downstream primers covering the mutation sites and perform whole-plasmid PCR. The primer details are shown in Table 3:

[0081] Table 3 Primers for single-site saturation mutagenesis

[0082]

[0083]

[0084] Table 4 PCR amplification system

[0085] Reagent Volume (μL) 2×Buffer 25 Plasmid 0.5 dNTP Mix 1 10 μmol / L Primer F 2 10 μmol / L Primer R 2 Enzyme 1 ddH2O 18.5 Total 50

[0086] Table 5 PCR amplification conditions

[0087]

[0088] Step 2 - Template digestion

[0089] Perform agarose gel electrophoresis on the PCR product, and after recovery, digest the plasmid template in it with DpnⅠ enzyme.

[0090] Table 6 Template digestion system

[0091]

[0092] Step 3 - Plasmid transformation

[0093] (1) Take out BL21(DE3) competent cells from the ultra-low temperature freezer (-80), place them on ice to melt, add 2 μL of plasmid to the competent cells and mix well, and incubate on ice for 30 min;

[0094] (2) Heat shock in a 42 °C water bath for 90 s and then quickly take it out and place it on ice for 5 min;

[0095] (3) Add 1 mL of antibiotic-free LB liquid medium and incubate on a shaker at 37 °C and 200 rpm for 1 h;

[0096] (4) After the cultivation, centrifuge at 4000 rpm for 5 min at room temperature;

[0097] (5) Discard 900 μL of the supernatant, mix the remaining supernatant with the precipitate, and spread it on an LB plate with the corresponding resistance;

[0098] (6) Incubate the above plate upside down in an incubator at 37 °C for 12 - 16 h to obtain single colonies.

[0099] Step 4 - Primary Screening

[0100] (1) First - level plate: Add 200 μL of LB medium containing 100 μg / mL kanamycin to a sterilized 96 - well deep - well plate, pick 3 single colonies into the 96 - well deep - well plate, and incubate it at 37 °C and 220 rpm for 8 h;

[0101] (2) Second - level plate: Add 400 μL of LB medium containing 100 μg / mL kanamycin to a sterilized 96 - well deep - well plate, pipette 50 μL of the bacterial solution from the first - level plate into the second - level plate, and place it at 37 °C

[0102] Shake - culture for 2 h;

[0103] (3) After taking the bacterial solution, add glycerol with a final concentration of 20% to the first - level plate and store it in a - 80 °C refrigerator for long - term preservation;

[0104] (4) Add IPTG with a final concentration of 0.4 mM to the second - level plate after 2 h of cultivation in (2), place it at 25 °C and 220 rpm, and continue the induction culture for 16 h;

[0105] (5) Centrifuge the above second - level plate at 4000 rpm and 4 °C for 20 min to collect the cells, and then place it at

[0106] -80 °C overnight;

[0107] (6) Take out the above second - level plate from - 80 °C, let it thaw at room temperature for 0.5 h, then add 300 μL of lysing solution (10 mM phosphate buffer at pH 7.5, 750 mg / L lysozyme, 10 mg / L DNaseⅠ) to each well, shake to suspend the cells, and place it on a shaker at 37 °C and 220 rpm for 1 h.

[0108] (7) After the incubation, centrifuge at 4000 rpm and 4 °C for 20 min, and take the supernatant for enzyme activity assay.

[0109] Step 5 - Re - screening

[0110] Use HPLC to re - screen the mutants that showed a significant increase in enzyme activity in the primary screening.

[0111] (1) Streak and activate the corresponding mutant strain plate of the first-level plate, pick a single colony and inoculate it into 5 ml of LB liquid medium containing 100 μg / mL kanamycin, and culture it with shaking at 37 °C for 12 h;

[0112] (2) Transfer it to 50 ml of LB liquid medium containing 100 μg / mL kanamycin according to an inoculation amount of 2%, and culture it with shaking at 37 °C until the OD 600nm reaches 0.6 - 0.8;

[0113] (3) Add IPTG with a final concentration of 0.4 mM, and induce culture at 25 °C for 16 h;

[0114] (4) After the culture is completed, centrifuge the culture solution at 4000×g at 4 °C for 10 min, discard the supernatant, and collect the cells. Wash the collected cells twice with 100 mM phosphate buffer at pH 7.5, resuspend them in the phosphate buffer, and ultrasonically disrupt them 30 times at a power of 400 W, with each ultrasonic treatment lasting for 3 s and an interval of 7 s. Centrifuge this cell lysate at 12000×g at 4 °C for 10 min to remove the precipitate, and the supernatant is the crude enzyme solution.

[0115] Step 6 - Enzyme activity assay of recombinant mutant NMNAT

[0116] Using NMN as the substrate, detect the enzyme activity of recombinant NMNAT according to the detection system in Table 7.

[0117] Table 7 Enzyme activity assay system

[0118] Reagent Name Volume (μL) 100 mmol / L Phosphate Buffer (pH 7.5) 760 300 mmol / L NMN 100 50 mmol / L ATP 100 <![CDATA[40mmol / L MgCl2]]> 20 Crude NMNAT Enzyme Solution 20 Final Volume 1000

[0119] React with shaking at 35 °C for 1 h, and terminate the reaction in a boiling water bath for 5 min. Centrifuge the reaction mixture at 5000 rpm for 5 min to remove cells and enzyme proteins. Use high-performance liquid chromatography to determine the NAD + generated in the reaction system. Enzyme activity definition: At 35 °C, every 1 g of the culture solution generates 1 μmol of NAD + per minute in the reaction system is defined as one enzyme activity unit. Determine the activity of all positive mutant NMNATs.

[0120] Step 7 - Detection of soluble expression of NMNAT genetic engineering bacteria

[0121] Unify the OD of the cell lysate of the genetic engineering bacteria after ultrasonic disruption in step 5 600After centrifugation at 12,000 rpm for 10 min at 4 °C for [[ID=]], 40 μL of the supernatant was taken and mixed with 10 μL of 5×SDS-PAGE Loading Buffer; the supernatant was discarded, and the precipitate was resuspended with the same volume of buffer. Then, 40 μL of the resuspended solution was taken and mixed with 10 μL of 5×SDS-PAGE Loading Buffer. High-throughput screening of 14 sites of NMNAT was carried out according to Steps 1-4, and 8 positive mutants were obtained. Enzyme activity verification was carried out on them according to Steps 5 and 6, and the results are shown in Table 8:

[0122] Table 8 Screening results of positive mutants in the NMNAT saturation mutation library

[0123] Mutant Name Mutation Site Codon Relative Enzyme Activity (%) WT - - 100 NMNAT-1 A62T ACA 124 NMNAT-2 A62D GAC 55 NMNAT-3 L78T ACC 116 NMNAT-4 V108A GCA 86 NMNAT-5 V108L CTC 29 NMNAT-6 F125Y TAC 163 NMNAT-7 K155E GAA 154 NMNAT-8 K155G GGA 102

[0124] As can be seen from the results in Table 8, there are 8 positive mutations in the NMNAT mutant strains. Among them, NMNAT-1, NMNAT-6, and NMNAT-7 showed excellent performance, which were 1.2 times, 1.6 times, and 1.5 times the enzyme activity of the wild type, respectively.

[0125] Example 3 NMNAT combinatorial mutation

[0126] According to the detection results of the mutants in Example 2, site-directed mutations were designed for the mutation sites A62T, F125Y, and K155E with excellent enzyme activity, and combinatorial mutants with multiple sites were constructed. The combinatorial mutants were screened according to the detection method in Example 2, and the results are shown in Table 9:

[0127] Table 9 Enzyme activity screening results of NMNAT combinatorial mutants

[0128]

[0129]

[0130] According to the screening results in Table 9, among the four mutant combinations, NMMAT-12 showed excellent performance. Compared with the wild type, the enzyme activity was greatly improved, which was 2.5 times the enzyme activity of the wild-type NMNAT.

[0131] Example 4 Transformation of NMNAT-WT

[0132] Using NMNAT as the catalytic enzyme, NAD was prepared. A four-necked round-bottom flask was used as the reaction vessel, and the reaction system was 100 mL, containing 4 g of NMN, 6.68 g of ATP, and 0.1 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 100 mL with phosphate buffer. 10 g of crude NMNAT enzyme solution and 10 g of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C under a water bath with a speed of 300 rpm. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 39.4% and the NMN conversion rate was 43.1% at 6 hours of reaction.

[0133] Example 5: Transformation of NMNAT Mutant

[0134] Using the NMNAT mutant NMNAT-6 as the catalytic enzyme, NAD was prepared. A four-necked round-bottom flask was used as the reaction vessel, and the reaction system was 100 mL, containing 4 g of NMN, 6.68 g of ATP, and 0.1 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 100 mL with phosphate buffer. 10 g of crude NMNAT enzyme solution and 10 g of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C under a water bath with a speed of 300 rpm. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 88.58% and the NMN conversion rate was 92.37% at 6 hours of reaction.

[0135] Example 6: Transformation of NMNAT Mutant Combinations

[0136] Using the NMNAT mutant combination NMNAT-9 as the catalytic enzyme, NAD was prepared. A four-necked round-bottom flask was used as the reaction vessel, and the reaction system was 100 mL, containing 4 g of NMN, 6.68 g of ATP, and 0.1 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 100 mL with phosphate buffer. 10 g of crude NMNAT enzyme solution and 10 g of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C under a water bath with a speed of 300 rpm. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 95.07% and the NMN conversion rate was 95.43% at 6 hours of reaction.

[0137] Example 7: Transformation of NMNAT Mutant Combinations

[0138] Using the NMNAT mutant combination NMNAT-12 as the catalytic enzyme, NAD was prepared. A four-neck round-bottom flask was used as the reaction vessel, and the reaction system was 100 mL, containing 4 g of NMN, 6.68 g of ATP, and 0.1 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 100 mL with phosphate buffer. 10 g of crude NMNAT enzyme solution and 10 g of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C and 300 rpm under water bath control. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 99.54% and the NMN conversion rate was 99.26% at 6 h of reaction.

[0139] Example 8 Transformation of NMNAT Mutant Combination

[0140] Using the NMNAT mutant combination NMNAT-12 as the catalytic enzyme, NAD was prepared. A four-neck round-bottom flask was used as the reaction vessel, and the reaction system was 30 L, containing 1.2 Kg of NMN, 2 Kg of ATP, and 30 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 30 L with water. 3 Kg of crude NMNAT enzyme solution and 3 Kg of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C and 300 rpm under water bath control. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 98.76% and the NMN conversion rate was 99.04% at 6 h of reaction.

[0141] Example 9 Transformation of NMNAT Mutant Combination

[0142] Using the NMNAT mutant combination NMNAT-12 as the catalytic enzyme, NAD was prepared. A 50 °C constant temperature reaction tank was used as the reaction vessel, and the reaction system was 30 L, containing 2 g of NMN, 3.34 g of ATP, and 0.1 g of magnesium chloride hexahydrate. The pH of the system was adjusted to 7.5 with ammonia water, and the volume was made up to 100 mL with phosphate buffer. 10 g of crude NMNAT enzyme solution and 10 g of crude pyrophosphatase enzyme solution were added. The reaction was stirred at 37 °C and 300 rpm under water bath control. During the reaction, the pH was controlled at about 7.5 by dropwise addition of ammonia water, and the reaction time was 6 hours. After the reaction, a sample was taken for HPLC detection. The results showed that the yield was 98.79% and the NMN conversion rate was 98.43% at 6 h of reaction.

[0143] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Mutants of NMNAT enzyme, The NMNAT enzyme is from Methanosphaera stadtmana; The mutation site of the mutant includes at least one of the 62nd, 125th and 155th positions.

2. The mutant according to claim 1, characterized in that The mutation site includes at least one of the following mutation sites: A62T, A62D, L78T, V108A, V108L, F125Y, K155E or K155G.

3. The mutant according to claim 1, characterized in that The mutation sites include A62T and F125Y; or including A62T and K155E; Or including A62T, F125Y and K155E.

4. A biological material comprising at least one of the following: A), encoding the mutant nucleic acid according to any one of claims 1 to 3; B), an expression unit comprising a promoter and the nucleic acid described in A); C), a plasmid vector comprising the nucleic acid described in A) and / or the expression unit described in B); D), containing the plasmid vector described in C), or a transformant having the nucleic acid described in A) and / or the expression unit described in B) integrated into its genome.

5. The biomaterial according to claim 4, characterized in that The nucleic acid sequence encoding the NMNAT enzyme is shown in SEQ ID NO.2, and in the mutant: The codon for the A62T site is ACA; the codon for the A62D site is GAC; the codon for the L78T site is ACC; the codon for the V108A site is GCA; the codon for the V108L site is CTC; the codon for the F125Y site is TAC; The codon at the K155E site is GAA; The codon for the K155G site is GGA.

6. The biomaterial according to claim 4, characterized in that The promoter in the expression unit is The backbone vector of the plasmid vector is pET-28a(+); The host of the transformant is Escherichia coli.

7. A method for preparing the mutant according to any one of claims 1 to 3, comprising culturing the transformant in the biological material according to any one of claims 4 to 6 to obtain a product containing the mutant according to any one of claims 1 to 3.

8. Use of the mutant according to any one of claims 1 to 3, the biomaterial according to any one of claims 4 to 6, or the product obtained by the preparation method according to claim 7 in the preparation of NAD+.

9. A method for preparing NAD+, characterized in that: Using NMN as a substrate and the mutant according to any one of claims 1 to 3 as a catalyst, a product containing NAD+ is prepared.

10. The preparation method according to claim 9, characterized in that: The pH value of the reaction system is 7-7.5, wherein the concentration of NMN is 0.12 mol / L; the reaction temperature is 37° C., and the reaction time is 6 h.

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