A thermostable nicotinamide phosphoribosyltransferase mutant and its application

Abstract

This invention provides a highly thermostable nicotinamide phosphoribosyltransferase mutant and its applications. The mutant was obtained through site-directed gene mutagenesis, and its amino acid sequence is shown in SEQ ID NO:4, and its nucleotide sequence is shown in SEQ ID NO:3. The mutant exhibits an 8.5-fold longer half-life compared to the parent protein. Under the same reaction conditions, the mutant demonstrates a higher catalytic production capacity for NMN than the parent protein, extending the production time. Furthermore, the mutant protein is immobilized, and the immobilized enzyme is applied in the process for preparing nicotinamide mononucleotides, improving enzyme reusability and operability, reducing enzyme preparation costs, and demonstrating high market competitiveness and industrial application value.

Description

Technical Field

[0001] This invention relates to the fields of molecular biology and enzyme engineering, and in particular to nicotinamide phosphoribosyltransferase mutants and their application in the synthesis of β-nicotinamide mononucleotides. Background Technology

[0002] Nicotinamide mononucleotide (NMN) is an important intermediate metabolite in the nicotinamide adenine dinucleotide (NAD) metabolic pathway. In the body, NMN is converted into NAD+, enabling it to exert its physiological functions, such as activating type III lysine deacetylases (Sirtuins) that use NAD+ as their sole substrate, maintaining cellular redox status, and regulating apoptosis. Studies have found that regulating NMN levels in the body can have a good preventive, therapeutic, and restorative effect on cardiovascular and cerebrovascular diseases, neurodegenerative diseases, and age-related degenerative diseases.

[0003] Due to its good therapeutic and restorative effects in delaying aging and repairing brain damage, and the latest research showing that it may prevent lung damage from air pollutants, β-nicotinamide mononucleotide (NMN) has received more attention and the market demand for it is increasing day by day.

[0004] There are three main existing methods for producing NMN: chemical synthesis, microbial fermentation, and in vitro enzymatic catalysis. Chemical synthesis is the earliest method, and several chemical synthesis methods for NMN have been patented. However, it suffers from problems such as difficulty in chiral resolution and the potential for harmful residues from organic solvents, leading to its gradual market obsolescence. Biosynthesis has become the industry trend for NMN production. Microbial fermentation and in vitro enzymatic catalysis can both be classified as biosynthesis, but fermentation is difficult to regulate metabolic pathways in vivo, resulting in low production efficiency and hindering industrial-scale production. In vitro enzymatic synthesis is currently the most ideal method for NMN production. Most existing in vitro enzymatic synthesis methods require the participation of nicotinamide phosphoribosyltransferase (Nampt, EC 2.4.2.12), which catalyzes the production of NMN using 5'-phosphoribose pyrophosphate (PRPP) and nicotinamide (NAM) as substrates. In the application of in vitro catalytic reactions, thermal stability is a key factor in improving the operability of an enzyme protein. Currently, the thermal stability of existing Nampts is poor, significantly increasing the production cost and severely impacting NMN yield. Against this backdrop, the modification of Nampt mutants with high thermal stability and their application in the catalytic synthesis of NMN, along with the improvement of their reusability and operability through immobilization technology, is of great significance for reducing the production cost of NMN and promoting the industrialization of NMN in vitro enzymatic catalytic synthesis. Summary of the Invention

[0005] To address the issues of low stability and poor industrial production capacity of existing nicotinamide phosphoribosyltransferases, the present invention aims to provide a nicotinamide phosphoribosyltransferase mutant with high thermal stability.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A highly thermally stable nicotinamide phosphoribosyltransferase mutant was developed with a single-point mutation at position 359, using the amino acid sequence of the parental nicotinamide phosphoribosyltransferase as a reference sequence.

[0008] The parent in this invention refers to nicotinamide phosphoribosyltransferase from Sulfurovum sp. FS06-10, whose nucleotide sequence is shown in SEQ ID NO.1 and amino acid sequence is shown in SEQ ID NO.2. The mutant, compared to the amino acid sequence shown in SEQ ID NO.2, contains a mutation at position 359 (Ala) to Phe, whose nucleotide sequence is shown in SEQ ID NO.3 and amino acid sequence is shown in SEQ ID NO.4.

[0009] The application of the nicotinamide phosphoribosyltransferase mutant in the catalytic synthesis of NMN. The nicotinamide phosphoribosyltransferase mutant can be used in the process of preparing nicotinamide mononucleotide, which can be a bio-enzymatic catalytic process, a microbial fermentation process, or a whole-cell catalytic process. Alternatively, the nicotinamide phosphoribosyltransferase mutant can be prepared as an immobilized enzyme, and the immobilized enzyme can be applied to the nicotinamide mononucleotide process.

[0010] Preferably, the temperature of the catalytic reaction is 50±10℃.

[0011] Preferably, the catalytic reaction temperature is 50±5℃.

[0012] The aforementioned bio-enzyme catalytic process for preparing NMN specifically refers to the process of converting substrates NAM and PRPP into NMN using bio-enzymes. The bio-enzyme is the nicotinamide phosphoribosyltransferase mutant of the present invention, or a combination of the nicotinamide phosphoribosyltransferase mutant of the present invention and one or more other enzymes. It can also be an immobilized enzyme prepared from the mutant of the present invention. The substrate can be PRPP and nicotinamide, or a precursor substance that can be converted into PRPP or nicotinamide. For example, using nicotinamide, ATP, and ribose as raw materials, NMN is prepared under the catalytic action of the nicotinamide phosphoribosyltransferase mutant of the present invention, ribophosphate pyrophosphate kinase, and ribokinase.

[0013] Preferably, the mutant is used in the form of enzyme solution, enzyme lyophilized powder, enzyme-containing cells, immobilized enzyme, or immobilized enzyme-containing cells.

[0014] The beneficial effects of this invention are as follows: This invention provides a nicotinamide phosphoribosyltransferase mutant. By performing site-directed mutagenesis on the nicotinamide phosphoribosyltransferase gene sequence, a highly stable nicotinamide phosphoribosyltransferase mutant is finally obtained. This mutant can efficiently catalyze the conversion of nicotinamide and phosphoribosyl pyrophosphate into NMN, and can be further prepared into an immobilized enzyme so that it can be reused, greatly reducing the production cost in the in vitro enzyme-catalyzed synthesis process of NMN, and has high industrial application value. Attached Figure Description

[0015] Figure 1 This is a graph showing the residual activity of the nicotinamide phosphoribosyltransferase mutant and its parent protein under different incubation times at 45°C.

[0016] Figure 2 This is a graph showing the results of NMN production catalyzed by the A359F single-point mutant protein and the parent protein under the same reaction conditions.

[0017] Figure 3 This is a graph showing the results of NMN production catalyzed before and after immobilization of the A359F single-point mutant protein.

[0018] Figure 4 It shows the number of times the immobilized enzyme has been reused and the residual enzyme activity. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed according to conventional experimental conditions or experimental conditions recommended by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention are commercially available.

[0020] Example 1: Construction of parental SuNampt protein expression strain

[0021] (1) The DNA sequence of Nampt protein from Sulfurovum sp.FS06-10 published in the database (NCBI: KIM05844.1) was used to synthesize the whole gene by a gene synthesis company. The synthesized DNA fragment was inserted between the NdeI and XhoI restriction sites of the pET28a plasmid to obtain the plasmid vector pET28a-SuNampt(WT) that expresses the parental Nampt protein.

[0022] (2) pET28a-SuNampt(WT) was transformed into BL21(DE3) competent cells (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.), and positive transformants were screened on LBK kanamycin resistant plates (i.e. LB solid plates containing 50 mg / L kanamycin, the same below). The correctly identified transformants were the expression strains expressing the parental SuNampt protein.

[0023] Example 2: Construction of a single-site mutant strain expressing the SuNampt protein

[0024] (1) Using the software Snapgene, corresponding site-directed mutagenesis primers were designed based on the SuNampt gene sequence synthesized in Example 1 (see Table 1 for specific primer sequences).

[0025] Table 1 Primer sequences for site-directed mutagenesis

[0026]

[0027] Using the plasmid vector pET28a-SuNampt(WT) from Example 1 as a template, PCR amplification was performed using KOD Fx Neo high-fidelity DNA polymerase (Toyobo Shanghai Biotechnology Co., Ltd.) with different primers. The PCR system is as follows:

[0028]

[0029] PCR products were digested using DpnI enzyme, and 10 μL of the digested product was transformed into *E. coli* Top10 competent cells (purchased from Beijing Zhuangmeng International Biotechnology Co., Ltd.). The cells were then plated on LBK kanamycin-resistant plates for positive transformant selection. Transformants correctly identified by colony PCR were then inoculated into LB medium, and plasmids were extracted. Sequencing by Shanghai Sangon Biotech Co., Ltd. confirmed the correct expression of the A359F mutant pET28a-SuNampt (A359F).

[0030] (2) The above expression plasmids were transformed into BL21(DE3) competent cells, and positive transformants were screened on LBK kanamycin resistant plates. The transformants that were correctly identified by colony PCR were the expression strains that expressed the single-site mutant Nampt protein.

[0031] Example 3: Expression and purification of SuNampt protein

[0032] (1) The parental SuNampt in Example 1 and the point mutant protein expression strain in Example 2 were inoculated into LBK medium and cultured for 16 h. Then they were transferred to LB medium and cultured until the OD600 value was 0.6-0.8. Then 0.2 mM IPTG was added to induce incubation and cultured overnight at 16°C.

[0033] (2) Centrifuge the above bacterial solution at 6000 rpm for five minutes and collect the bacterial cells.

[0034] (3) Resuspend the bacterial cells in Tris-HCl buffer and break them up using an ultrasonic disruptor.

[0035] (4) Centrifuge the above-mentioned broken liquid at 12,000 rpm for 30 minutes at 4℃ and collect the supernatant.

[0036] (5) Purify the supernatant using a nickel column. First, wash the nickel column with 50mM imidazole Tris-HCl buffer to elute impurities, then wash the nickel column with 200mM imidazole Tris-HCl buffer to elute Nampt. Collect the eluent to obtain the purified SuNampt protein.

[0037] Example 4: Evaluation of the thermal stability of the mutant

[0038] (1) Take 100 μL of the purified SuNampt enzyme solution from Example 3 into a 1.5 mL centrifuge tube and incubate it in a constant temperature mixer at 45 °C for different times. After incubation, centrifuge at 12000 rpm for 2 min to separate the precipitate. Then, take the supernatant from the centrifugation to prepare a 100 μL enzyme activity assay reaction system: 2 mM NAM, 0.75 mM PRPP, 2.5 mM MgCl2, 8 mM ATP, 1 μM purified SuNampt protein, and 50 mM Tris-HCl (pH 7.5). Place the prepared reaction system in a constant temperature mixer at 50 °C and incubate for 15 min. After the reaction, determine the concentration of NMN generated by HPLC and calculate the enzyme activity. Use the activity of the enzyme solution that has not undergone heat incubation as a control to calculate the remaining enzyme activity.

[0039] Compared to the parental SuNampt protein, the A359F single-point mutant protein exhibits significantly improved thermostability. The parental protein essentially loses all activity after incubation at 45°C for 1 hour, while the mutant protein retains 84% ​​of its catalytic activity under the same treatment conditions. Figure 1 Half-life t 1 / 2 It is approximately 8.5 times higher than the parent protein. It can meet the application requirements of in vitro multi-enzyme synthesis systems under most conditions. Figure 1 This indicates that we have successfully modified and obtained a nicotinamide phosphoribosyltransferase mutant with high thermal stability.

[0040] Example 5: Evaluation of the mutant's ability to produce NMN

[0041] A 100 μL initial reaction mixture was prepared in a 1.5 mL centrifuge tube containing: 9 mM NAM, 2 mM PRPP, 20 mM MgCl2, 24 mM ATP, 1 μM purified SuNampt protein, 0.04 g / L pyrophosphate hydrolase, and 250 mM Tris-HCl (pH 7.5). The SuNampt protein was either the A359F single-point mutant protein purified in Example 4 or the SuNampt parent protein, and the pyrophosphate hydrolase was commercially available enzyme powder. The prepared reaction mixture was placed in a 50°C incubator and allowed to react for different times. Every hour after 1 hour of reaction, PRPP was added to the reaction mixture to a final concentration of 0.5 mM. The concentration of NMN generated was determined by HPLC after the reaction reached the sampling point.

[0042] The results showed that at 1 hour of reaction, the NMN yields catalyzed by the A359F single-point mutant protein and the SuNampt parental protein were similar. However, after 1 hour, the SuNampt parental protein was essentially inactivated, and the NMN yield in the NMN reaction system no longer increased. The A359F single-point mutant protein maintained good catalytic activity from 1 to 5 hours of reaction. At 5 hours, the NMN yield was 1.33 mM, corresponding to a PRPP conversion rate of 32.5%. The parental SuNampt protein, after 5 hours of reaction, produced 0.77 mM of NMN, corresponding to a PRPP conversion rate of 19%. Figure 2 The above results indicate that the modified nicotinamide phosphoribosyltransferase mutant has a better ability to catalyze the production of NMN than the parent protein, and is more in line with the application requirements of in vitro multi-enzyme synthesis systems.

[0043] Example 6: Immobilized enzyme-catalyzed production of NMN

[0044] The A359F single-point mutant protein was immobilized on LX-1000EP resin, and an immobilized enzyme was used to catalyze the reaction. The immobilized enzyme was prepared as follows: The purified SuNampt protein obtained in Example 3 was replaced with 20mM phosphate buffer. 1g of LX-1000EP resin, 2.5mg of enzyme protein, and 5ml of 20mM phosphate buffer were added to a stoppered shake flask and incubated at 250rpm for 24h in a constant temperature shaker at 25℃ to obtain the immobilized enzyme. The specific reaction system was as follows: 2mM nicotinamide (NAM), 8mM ATP, 2.5mM MgCl2, 0.75mM PRPP, 1μM purified SuNampt protein, and 20mM phosphate buffer. The SuNampt protein was either the A359F single-point mutant SuNampt protein purified in Example 3 or the immobilized SuNampt protein in Example 6. The reaction system was controlled at pH 8, and the reaction was carried out at 50℃ for 15 min. The NMN yield was analyzed by HPLC. The results showed that the production efficiency of NMN using immobilized enzymes and free enzymes was comparable, with NMN conversion rates of 52% and 48% for PRPP, respectively. Figure 3 ).

[0045] Example 7: Reuse of Immobilized Enzymes

[0046] The immobilized enzyme obtained in Example 6 was used in NMN production. After the NMN production reaction was completed, the immobilized enzyme was recovered by filtration, rinsed twice with deionized water, and then used again in the in vitro enzyme reaction, using the same reaction system as in Example 6. The enzyme activity after different uses was compared with the enzyme activity after the first use to examine the change in residual activity with the number of uses. The results are shown below. Figure 4 It can be seen that the immobilized enzyme activity decreases relatively quickly, and further improvements are needed.

[0047] Based on the results in Examples 1-7, it is demonstrated that the present invention successfully modified and obtained a nicotinamide phosphoribosyltransferase mutant with high thermal stability. The mutant protein has a higher NMN production capacity than the parent protein. Immobilized enzyme reduces enzyme preparation costs and has high market competitiveness and industrial application value.

[0048] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A highly thermally stable nicotinamide phosphoribosyltransferase mutant, characterized in that, The amino acid sequence of the mutant is shown in SEQ ID NO:

4.

2. The gene encoding the mutant of claim 1, the nucleotide sequence of which is shown in SEQ ID NO:

3.

3. The application of the nicotinamide phosphoribosyltransferase mutant of claim 1 in the catalytic synthesis of NMN.

4. The application according to claim 3, characterized in that, The temperature of the catalytic reaction is 50±10℃.

5. The application according to claim 4, characterized in that, The catalytic reaction temperature is 50±5℃.

6. The application according to claim 3, 4, or 5, characterized in that, The substrates for the catalytic reaction are PRPP and NAM, or precursors that can be converted into PRPP or nicotinamide.

7. The application according to claim 6, characterized in that, The mutant, in combination with ribophosphate pyrophosphate kinase and ribokinase, catalyzes a reaction to generate NMN.

8. The application according to claim 7, characterized in that, The mutants are used in the form of enzyme solution, enzyme lyophilized powder, enzyme-containing cells, or immobilized enzymes.

9. The application according to claim 8, characterized in that, The mutant is used in the form of immobilized enzyme-containing cells.

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