A nicotinamide phosphoribosyltransferase mutant and its application

Abstract

The present invention provides a nicotinamide phosphoribosyltransferase mutant and its application, relating to the field of bioengineering technology. The nicotinamide phosphoribosyltransferase mutant comprises a protein having one or more mutations at the following positions relative to the reference sequence: position 190, position 308, position 348, position 387, position 241, wherein the amino acid position numbers are defined by the reference sequence, and the reference sequence is as shown in SEQ ID No.1. In this application, it is demonstrated that mutating the amino acid at position 190 of nicotinamide phosphoribosyltransferase from a phenylalanine residue to a tryptophan residue can effectively improve the relative enzyme activity, and applying it to the process of catalytically generating nicotinamide mononucleotide can effectively improve the yield of nicotinamide mononucleotide, providing a new, purifiable, highly catalytically active and highly efficient engineering enzyme for the production of nicotinamide mononucleotide.

Description

Technical Field

[0001] This application relates to the field of bioengineering technology, and particularly relates to a nicotinamide phosphoribosyltransferase mutant and its application. Background Art

[0002] Nicotinamide adenine dinucleotide (NAD) is a coenzyme that widely exists in organisms and transfers electrons and reducing hydrogen. It exists as a cofactor and substrate in multiple biocatalytic reactions. In recent years, with the gradual in-depth research, more data shows that the decrease in NAD+ level will lead to changes in cell metabolic function and an increase in disease susceptibility, while restoring the NAD+ level can prevent the occurrence and development of diseases. Since NAD cannot increase its own content by direct supplementation, supplementing its precursor has become the main way to supplement NAD. As a direct precursor of NAD, nicotinamide mononucleotide (NMN) plays an important role in increasing the NAD content in cells. NMN is rapidly absorbed in the body, can quickly increase the NAD content in cells, improve the survival rate of nerve cells, reduce cell apoptosis, has a good supplementation effect, and has no obvious effect on the activity and function of various enzymes in the synthesis pathway, and has certain biological safety. It has good application value in age-related aging degenerative diseases, neurodegenerative diseases, metabolic diseases, anti-aging, etc.

[0003] Biocatalytic synthesis of NMN has become a research hotspot for various biomedical companies, and among them, nicotinamide phosphoribosyltransferase (Nampt) is the most critical rate-limiting enzyme in the enzyme reaction. In the currently published literature, the main species sources of Nampt are Meiothermus ruber DSM 1279 (CN 108026517B), homo sapien (Wang Feng et al., "Journal of Zhejiang University (Medical Sciences)", 2011, Vol. 40), Luteibacter sp. (CN 110373397A), Methanobacteriumsp.PtaU1.Bin097 (CN 113106080 A), etc. However, the wild-type Nampt of these species still has problems such as low catalytic activity, high production cost, and inability to be purified. Summary of the Invention

[0004] Liao Yibo et al. constructed Nampt from three species, namely human, Meiothermus ruber, and Comamonadaceae bacterium, in the article "Biosynthesis of Nicotinamide Mononucleotide by Enzymatic Method", and measured their activities respectively. The results showed that Nampt from Comamonadaceae bacterium had the highest activity among the reported literatures (kcat / Km was about 2.7 times that of Nampt from Meiothermus ruber). In this application, on this basis, the sequence of Nampt from Comamonadaceae bacterium was further modified and optimized to construct a Nampt mutant that could be purified and had higher catalytic activity.

[0005] On the one hand, this application provides a nicotinamide phosphoribosyltransferase mutant, and the nicotinamide phosphoribosyltransferase mutant includes any one of the following A1)-A3):

[0006] A1) A protein containing one or more mutations at the following positions relative to the reference sequence: position 190, position 308, position 348, position 387, position 241, wherein the amino acid position numbers are defined by the reference sequence, and the reference sequence is as shown in SEQ ID No.1;

[0007] A2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues from the amino acid sequence of the protein described in A1), having more than 90% identity with the protein described in A1) and having the same function;

[0008] A3) A fusion protein obtained by connecting a tag to the N (nitrogen) - terminus and / or C (carbon) - terminus of A1) or A2) and having the same function.

[0009] Preferably, the reference sequence is the amino acid sequence of Nampt from Comamonadaceae bacterium, and the NCBI accession number of Nampt is RYF34637.1. In a preferred case, a his x 6 tag is added to its N - terminus.

[0010] Preferably, the protein of A2) has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the amino acid sequence defined in A1).

[0011] More preferably, the nucleotide sequence encoding the reference sequence includes the nucleotide sequence as shown in SEQ ID NO.2 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.2.

[0012] More preferably, the coding sequence of the reference sequence (i.e., Nampt derived from Comamonadaceae bacterium) includes the nucleotide sequence shown in SEQ ID No.2 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity with SEQ ID NO.2.

[0013] Those skilled in the art can understand that the protein coding rules in this application follow common rules. Taking the 190th position as an example, the 190th mutation refers to the 190th amino acid site counted sequentially from the N (nitrogen) - terminal to the C (carbon) - terminal direction of the reference sequence.

[0014] In a preferred embodiment, the nicotinamide phosphoribosyltransferase mutant:

[0015] is obtained by mutating the phenylalanine residue (F) at the 190th position of the reference sequence to a tryptophan residue (W) or an alanine residue (A);

[0016] is obtained by mutating the aspartic acid residue (D) at the 308th position of the reference sequence to an alanine residue (A), an asparagine (N) or a glutamic acid (E);

[0017] is obtained by mutating the aspartic acid residue (D) at the 348th position of the reference sequence to an asparagine (N) or a glutamic acid (E);

[0018] is obtained by mutating the aspartic acid residue (D) at the 387th position of the reference sequence to an alanine residue (A) or a glutamic acid (E);

[0019] is obtained by mutating the glutamic acid (E) at the 241st position of the reference sequence to an alanine residue (A) or a glutamine (Q).

[0020] Furthermore, the nicotinamide phosphoribosyltransferase mutant is obtained by mutating the 190th position of the reference sequence; preferably, the phenylalanine residue (F) at the 190th position is mutated to a tryptophan residue (W) or an alanine residue (A); more preferably, the phenylalanine residue at the 190th position is mutated to a tryptophan residue.

[0021] Furthermore, the nicotinamide phosphoribosyltransferase mutant includes the amino acid sequence shown in SEQ ID NO.3; preferably, the nucleotide sequence encoding the nicotinamide phosphoribosyltransferase mutant includes the nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.4.

[0022] More preferably, the coding sequence of the nicotinamide phosphoribosyltransferase mutant comprises the nucleotide sequence shown in SEQ ID No. 4 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity with SEQ ID NO. 4.

[0023] It can be understood that those skilled in the art can select a suitable gene editing system and gene editing method according to the actual situation to complete the construction of the above mutants.

[0024] In a preferred embodiment, the mutant is obtained by whole plasmid PCR.

[0025] On the other hand, the present application also provides a biological material, which comprises any one of the following B1)-B6):

[0026] B1) A nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant;

[0027] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0028] B3) A recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2);

[0029] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3);

[0030] B5) A recombinant cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3);

[0031] B6) A whole cell catalyst containing the nucleic acid molecule described in B1), the expression cassette described in B2), the recombinant vector described in B3), the recombinant microorganism described in B4), and / or the recombinant cell described in B5).

[0032] Functional elements such as promoters, terminators, and marker genes may also be included in the expression cassette described herein. Those skilled in the art can make routine selections according to the actual situation as long as the expression of the nucleic acid molecule described in B1) can be completed. The structure and composition of the expression cassette will not be overly restricted herein.

[0033] The vector described in this article refers to a vector that can transport exogenous DNA or a target gene into a host cell for amplification and expression. The vector can be a cloning vector or an expression vector, including but not limited to: plasmids, phages (such as λ phage or M13 filamentous phage, etc.), cosmids (i.e., cosmid plasmids), or viral vectors. Specifically, it can be the vector pET30a plasmid.

[0034] The cells described in this article can be plant cells or animal cells, and the cells can be any biological cells that can synthesize the target nicotinamide mononucleotide.

[0035] Furthermore, the nucleic acid molecule includes a nucleotide sequence shown in SEQ ID NO.4 or a nucleotide sequence having at least 98% sequence identity with SEQ ID NO.4.

[0036] Preferably, the nucleic acid molecule includes the nucleotide sequence shown in SEQ ID No.4 or a nucleotide sequence having 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% sequence identity with SEQ ID NO.4.

[0037] Furthermore, the recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli, and Saccharomyces cerevisiae.

[0038] Preferably, the recombinant microorganism is Escherichia coli.

[0039] In a preferred embodiment, the Escherichia coli is Escherichia coli E.coli BL(21)DE3.

[0040] On the other hand, this application also provides the use of the described biological material in the preparation of nicotinamide phosphoribosyltransferase.

[0041] On the other hand, this application also provides the use of the nicotinamide phosphoribosyltransferase mutant or the described biological material in enhancing the activity of nicotinamide phosphoribosyltransferase.

[0042] On the other hand, this application also provides the use of the nicotinamide phosphoribosyltransferase mutant or the described biological material in the preparation of nicotinamide mononucleotide.

[0043] On the other hand, this application also provides a method for preparing the nicotinamide phosphoribosyltransferase mutant, and the method includes the following steps:

[0044] Step 1. Ferment and culture the biological material, and collect the precipitate by centrifugation; wherein, the biological material is a recombinant microorganism or a recombinant cell;

[0045] Step 2. Resuspend the precipitate, disrupt it by ultrasound, and centrifuge to obtain the supernatant, i.e., the protein solution;

[0046] Step 3. Elute and dialyze the protein solution to obtain the nicotinamide phosphoribosyltransferase mutant.

[0047] Preferably, the preparation method of the nicotinamide phosphoribosyltransferase mutant comprises the following steps:

[0048] Step 1. Culture the recombinant microorganism in a medium at 25°C - 40°C and 100 - 200 rpm for 5 - 12 h to obtain a seed solution, transfer it to a resistant fermentation medium at an inoculation amount of 1 - 5%, culture it at 25°C - 40°C and 100 - 200 rpm for 5 - 12 h, add IPTG with a final concentration of 0.1 - 0.5 mM, and induce and culture it at 10°C - 20°C for 12 - 24 h, then collect the cells by centrifugation;

[0049] Step 2. Resuspend the collected cells, add 1 - 5 mM PMSF, then disrupt the cells by ultrasound, centrifuge at 10000 - 15000 rpm for 5 - 15 min, and the obtained supernatant is the protein solution containing the nicotinamide phosphoribosyltransferase mutant;

[0050] Step 3. Elute the protein solution with an imidazole solution at 0°C - 4°C, and dialyze to remove imidazole to obtain the nicotinamide phosphoribosyltransferase mutant.

[0051] In a preferred embodiment, the purification method of the nicotinamide phosphoribosyltransferase mutant comprises the following steps:

[0052] Step 1. Culture the recombinant microorganism in an LB medium at 37°C and 180 rpm for 8 h to obtain a seed solution, transfer it to a resistant fermentation medium at an inoculation amount of 2%, culture it at 37°C and 140 rpm for 8 h, add IPTG with a final concentration of 0.2 mM, and induce and culture it at 16°C for 16 h, then collect the cells by centrifugation;

[0053] Step 2. Resuspend the collected cells with 25 mM Tris and 150 mM NaCl, add 1 mM PMSF, then disrupt the cells by ultrasound, centrifuge at 12000 rpm for 10 min, and the obtained supernatant is the protein solution containing the nicotinamide phosphoribosyltransferase mutant;

[0054] Step 3: Incubate the protein solution with His-tag Purification Resin at 4°C, elute with imidazole solution, and dialyze in 25 mM Tris, 150 mM NaCl solution to remove imidazole, obtaining the nicotinamide phosphoribosyltransferase mutant; the imidazole solution includes imidazole solutions with concentrations of 20 mM and 150 mM.

[0055] On the other hand, the present application also provides a method for preparing nicotinamide mononucleotide, the method comprising: adding the nicotinamide phosphoribosyltransferase mutant to a substrate solution for reaction to obtain nicotinamide mononucleotide.

[0056] Preferably, a method for preparing nicotinamide mononucleotide, the method comprising: adding the nicotinamide phosphoribosyltransferase mutant, ribose phosphate pyrophosphokinase, and ribokinase to a substrate solution for reaction, with a stirring speed of 100 - 400 rpm, a reaction temperature of 25°C - 40°C, a pH value of 7.0 - 7.5, and reacting for 1 - 5 h.

[0057] Preferably, the concentration of the nicotinamide phosphoribosyltransferase mutant is 0.1 - 0.5 g / L, the concentration of ribose phosphate pyrophosphokinase is 0.1 - 0.5 g / L, and the concentration of ribokinase is 0.1 - 0.5 g / L.

[0058] Preferably, the substrate includes 1 - 5 mM nicotinamide, 10 - 50 mM ATP, 1 - 5 mM ribose, 12 - 15 mM MgCl2, 10 - 50 mM Tris-HCl buffer, with a pH of 7.0 - 7.5.

[0059] Preferably, the method further includes steps of purification and drying.

[0060] In a preferred embodiment, a method for preparing nicotinamide mononucleotide includes: adding the nicotinamide phosphoribosyltransferase mutant, ribose phosphate pyrophosphokinase, and ribokinase to a substrate solution for reaction, with a stirring speed of 300 rpm, a reaction temperature of 37°C, a pH value of 7.0 - 7.5, and reacting for 4 h.

[0061] Preferably, the concentration of the nicotinamide phosphoribosyltransferase mutant is 0.1 g / L, the concentration of ribose phosphate pyrophosphokinase is 0.2 g / L, and the concentration of ribokinase is 0.1 g / L.

[0062] Preferably, the substrate includes 1 mM nicotinamide, 10 mM ATP, 1 mM ribose, 12 mM MgCl2, 50 mM Tris-HCl buffer, with a pH of 7.0 - 7.5.

[0063] The present invention has the following beneficial effects:

[0064] In this application, for the first time, the sequence of Nampt derived from Comamonadaceae bacterium was further modified and optimized. By performing single-site mutagenesis on Nampt, a beneficial mutation site at position 190 was obtained. It was demonstrated that mutating the 190th amino acid of Nampt from phenylalanine residue to tryptophan residue could effectively improve the relative enzyme activity, and applying it to the process of catalyzing the production of nicotinamide mononucleotide could effectively increase the yield of nicotinamide mononucleotide, providing a new, purifiable, highly catalytically active, and efficient engineering enzyme for the production of nicotinamide mononucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The drawings described herein are used to provide a further understanding of the present application and form a part of the present application. The schematic embodiments of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation of the present application. In the drawings:

[0066] Figure 1 is the purification result diagram of nicotinamide phosphoribosyltransferase Nampt;

[0067] Figure 2 is the chromatogram result diagram of nicotinamide mononucleotide NMN product. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0068] Identity: In molecular evolution research, it refers to the degree of similarity between the nucleotide sequences of two nucleic acid molecules or the amino acid sequences of two protein molecules.

[0069] Expression cassette: An expression cassette refers to a set of DNA sequences composed of a promoter, a target gene, a reporter gene, etc., which can be expressed in specific tissues and are easy to detect.

[0070] Recombination: Broadly speaking, any process of genetic exchange that causes genotype changes is called recombination.

[0071] Recombinant vector: A recombinant vector is a vector that transfers a target gene on the basis of the basic skeleton of a cloning vector, so that the target gene can be expressed.

[0072] Recombinant microorganism: A fungal cell line in which a foreign gene is highly expressed by genetic engineering methods.

[0073] Recombinant cell: The term "recombinant cell" means any cell type that is easily transformed, transfected, transduced, etc. with a nucleic acid construct or expression vector containing the polynucleotide of the present invention. The term "recombinant cell" encompasses any progeny of a parental cell that is not completely identical to the parental cell due to mutations occurring during replication.

[0074] Whole-cell catalyst: Whole-cell biocatalysis refers to the process of using intact biological organisms (i.e., whole cells, tissues, or even individuals) as catalysts for chemical transformation. The corresponding intact biological organisms participating in this catalytic process are whole-cell catalysts.

[0075] Inoculation: The inoculation amount refers to the ratio of the volume of the transferred seed culture to the volume of the culture after inoculation.

[0076] To more clearly illustrate the overall concept of this application, the following will be described in detail by way of examples in conjunction with the accompanying drawings of the specification. In the following description, a large number of specific details are given to provide a more thorough understanding of the present invention. However, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details. In other examples, in order to avoid confusion with the present invention, some well-known technical features in the art are not described.

[0077] For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer.

[0078] Unless otherwise specified, in the following embodiments, for reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained by commercial purchase.

[0079] In the following examples, plasmids, restriction enzymes, PCR enzymes, column DNA extraction kits, DNA gel recovery kits, etc. used are commercial products, and the specific operations are carried out according to the kit instructions.

[0080] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in this technical field, and can be specifically carried out according to Molecular Cloning: A Laboratory Manual (Fourth Edition).

[0081] In addition, the "water" mentioned in the present invention includes any feasible water that can be used in this field, such as deionized water, distilled water, ion-exchanged water, double-distilled water, high-purity water, pure water, etc.

[0082] Example 1 Construction of the parental nampt plasmid

[0083] The full - sequence optimization synthesis was performed on the gene sequence of the parental nicotinamide phosphoribosyltransferase from Comamonadaceae bacterium (NAMPT, NCBI accession number: RYF34637.1) published in the gene bank (completed by GenScript Biotech Corporation). The synthesized product was digested with restriction endonucleases NdeI and NotI and then ligated to the vector pET - 30a digested with the same restriction endonucleases NdeI and NotI to obtain plasmid pET30a - nampt. Through DNA sequencing, it was determined that the nucleotide sequence of the cloned parental nicotinamide phosphoribosyltransferase was as shown in SEQ ID NO: 2, and its amino acid sequence was as shown in SEQ ID NO: 1.

[0084] Example 2 Expression and purification of parental nampt protein

[0085] The constructed recombinant plasmid pET30a - nampt was transformed into BL21(DE3) competent cells and cultured overnight at 37°C in an inverted manner on an LB plate containing Kan resistance. Positive monoclonal cells were selected to obtain a genetically engineered strain of nicotinamide phosphoribosyltransferase (NAMPT) that could be induced to express.

[0086] The BL21(DE3) cells (genetically engineered strain) containing the NAMPT gene were inoculated into a 10 - mL centrifuge tube of LB medium containing Kan resistance and cultured in a shaker at 37°C and 180 rpm for 8 h to obtain a seed culture solution.

[0087] The seed culture solution was transferred to a 1 - L LB liquid medium containing Kan resistance at an inoculation ratio of 2% and placed in a shaker. It was cultured at 37°C and 140 rpm for 8 h, and OD 600 When it reached 0.6 - 0.8, the temperature was lowered to 16°C, IPTG was added, and the concentration was controlled at 0.2 mM for 16 h of induction expression. The fermentation broth was centrifuged at 4°C to collect the bacterial cells, and the collected bacterial cells were resuspended with 25 mM Tris, 150 mM NaCl (pH = 7.5). After adding 1 mM PMSF, the cells were broken by ultrasonic treatment. Then, it was centrifuged again at 12000 rpm for 10 min, and the obtained supernatant was a protein solution containing nampt (the supernatant was denoted as CS, and the precipitate was denoted as CP). The supernatant was co - incubated with a certain amount of His - tag Purification Resin in a rotary shaker at 4°C, and the effluent solution (denoted as FL) was eluted successively with a low - concentration imidazole solution (20 mM imidazole, 25 mM Tris, 150 mM NaCl, denoted as L) and a high - concentration imidazole solution (150 mM imidazole, 25 mM Tris, 150 mM NaCl, denoted as H), as shown in Table 1. The purification of the protein could be seen through electrophoresis, see attached Figure 1(Purity > 90%). The obtained protein solution was dialyzed at 4°C using a 3500 kD dialysis bag in a solution of 25 mM Tris and 150 mM NaCl (pH = 7.5) to remove imidazole. The obtained nampt can be used for enzyme activity assays and the preparation of NMN.

[0088] Table 1

[0089]

[0090] Construction of Mutants in Example 3

[0091] Site-directed mutagenesis was performed on the amino acids at positions 190, 308, 348, and 387 of the nampt obtained in Example 2 (counting sequentially from the nitrogen terminus to the carbon terminus of the amino acid sequence). Mutagenic primers were designed using Primer5 software, and mutants were obtained by whole plasmid PCR. The specific primer designs are shown in Table 2, with the Forward sequence above and the Reverse sequence below.

[0092] Using Primer STAR HS DNA polymerase (TaKaRa), with the participation of forward and reverse saturation primers, whole plasmid amplification reaction PCR was carried out using the recombinant plasmid pET30a-NAMPT as a template. The reaction program refers to the reagent instruction manual. 1 μL of restriction endonuclease Dpn I was added to the product, and after treatment at 37°C for 30 min, it was transferred into Dh5α competent cells and cultured overnight at 37°C inverted on an LB plate containing Kan resistance. Positive monoclonal cells were selected and sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for sequencing.

[0093] Table 2

[0094]

[0095]

[0096] Note: Among them, taking the mutant site F190A as an example, "F190A" means that the amino acid at the 190th site of nampt is mutated from F to A.

[0097] Expression and Purification of Mutant Enzymes in Example 4

[0098] The correctly sequenced Dh5α cells in Example 3 were extracted using a plasmid miniprep kit (Tiangen) to obtain mutant plasmids. Each mutant plasmid was transferred into BL21(DE3) competent cells, and the expression and purification of mutant proteins were carried out according to the method in Example 2.

[0099] Determination of Enzyme Activity in Example 5

[0100] Take 100 μg of the parents and each mutant protein from Example 2 and Example 4, and add them to 1 mM nicotinamide, 4 mM PRPP, 12 mM MgCl2, 4 mM ATP and 50 mM Tris buffer respectively, and adjust the pH to 7.5. React at 37 °C and 600 rpm for 15 min. The reaction was terminated by adding 25% trichloroacetic acid in a volume ratio of 9:1. The content of nicotinamide mononucleotide (NMN) in the reaction solution was determined by high performance liquid chromatography (HPLC), and the specific enzyme activity of each enzyme was calculated. Taking the specific enzyme activity of the parental nicotinamide phosphoribosyltransferase as a reference of 100, the relative specific activities of the parents and each mutant are shown in Table 5.

[0101] The calculation method of the yield includes: First, obtain the peak area (A) and retention time (t) of each compound from the chromatogram. Then, use the following formula to calculate the yield (Y) of each compound: Y = A × (t2 - t1) × 60 / (t3 - t2). Wherein, t1 and t2 are the start and end times of each compound peak, and t3 is the start time of the next compound peak in the chromatogram.

[0102] The calculation method of relative activity includes: Relative activity = (activity of the enzyme to be measured / activity of the standard enzyme) × 100%.

[0103] Table 5

[0104]

[0105]

[0106] As can be seen from the results in Table 5, it is proved in this example that the mutation of the 190th amino acid site from F to W is a beneficial mutation. Compared with the Nampt parent, the relative enzyme activity of its mutant has increased, and the yield of nicotinamide mononucleotide has also increased during the process of using this mutant to catalyze the formation of nicotinamide mononucleotide.

[0107] Example 6 Catalytic synthesis of nicotinamide mononucleotide by nicotinamide phosphoribosyltransferase mutant

[0108] Add the substrate solution (containing 1 mM nicotinamide, 10 mM ATP, 1 mM ribose, 12 mM MgCl2, and 50 mM Tris-HCl buffer) with a pH of 7.0 - 7.5 into the reaction tube. Then add various enzymes for catalysis, and the addition amounts of various enzymes are as follows: 0.1 g / L of the protein solution of the nicotinamide phosphoribosyltransferase mutant (F190W) obtained in Example 4, 0.2 g / L of ribose phosphate pyrophosphokinase, and 0.1 g / L of ribokinase. Stir continuously during the reaction (stirring speed 300 rpm), control the reaction temperature at 37 °C, maintain the pH value at 7.0 - 7.5, and after reacting for 4 h, a crude product solution of nicotinamide mononucleotide (containing 0.9 mM NMN) is obtained. After filtration, purification, and drying, the finished product of nicotinamide mononucleotide is obtained. The preparation of NMN can be seen by chromatography, see Appendix Figure 2 (purity > 90%).

[0109] The above are only the embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the scope of the claims of the present application.

Claims

1. A nicotinamide phosphoribosyltransferase mutant, characterized in that: The nicotinamide phosphoribosyltransferase mutant is obtained by mutation at position 190 of the reference sequence, wherein the phenylalanine residue at position 190 is mutated to a tryptophan residue, and the reference sequence is shown in SEQ ID No.

1.

2. Biomaterial, characterized in that The biological material includes any one of the following B1)-B6): B1) a nucleic acid molecule encoding the nicotinamide phosphoribosyltransferase mutant according to claim 1; B2) an expression cassette, the expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector, the recombinant vector containing the nucleic acid molecule described in B1) and / or the expression cassette described in B2); B4) a recombinant microorganism, wherein the recombinant microorganism contains the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B5) a recombinant cell, the recombinant cell containing the nucleic acid molecule described in B1), the expression cassette described in B2), and / or the recombinant vector described in B3); B6) A whole-cell catalyst, wherein the whole-cell catalyst comprises the nucleic acid molecule described in B1), the expression cassette described in B2), the recombinant vector described in B3), the recombinant microorganism described in B4, and / or the recombinant cell described in B5).

3. The biomaterial according to claim 2, characterized in that The recombinant microorganism is one or more of Corynebacterium glutamicum, Bacillus subtilis, Escherichia coli and Saccharomyces cerevisiae.

4. Use of the biomaterial according to claim 2 or 3 in the preparation of nicotinamide phosphoribosyltransferase.

5. A method for preparing the nicotinamide phosphoribosyltransferase mutant according to claim 1, characterized in that: The method comprises the following steps: Step 1: fermenting and culturing the biological material according to claim 2 or 3, and collecting the precipitate by centrifugation; wherein the biological material is a recombinant microorganism or a recombinant cell; Step 2, resuspending the precipitate, ultrasonically disrupting it, and centrifuging it to obtain a supernatant, i.e., a protein solution; Step 3: eluting and dialyzing the protein solution to obtain a nicotinamide phosphoribosyltransferase mutant.

6. Use of the nicotinamide phosphoribosyltransferase mutant according to claim 1 or the biomaterial according to claim 2 or 3 in the preparation of nicotinamide mononucleotide.

7. A method for preparing nicotinamide mononucleotide, characterized in that: The method comprises: adding the nicotinamide phosphoribosyltransferase mutant according to claim 1 to a substrate solution for reaction to obtain nicotinamide mononucleotide.

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