Glutamine transaminase mutants and their applications

Abstract

This invention discloses a glutamine transaminase mutant and its applications, belonging to the field of bioengineering technology. The glutamine transaminase mutant of this invention is obtained by mutation based on the wild-type glutamine transaminase shown in SEQ ID NO.1. This invention modifies the substrate spectrum of the glutamine transaminase derived from Streptomyces, and the obtained glutamine transaminase mutant can catalyze the synthesis of nicotinamide from nicotinic acid. Combined with the synthesis method of this invention, an extremely high nicotinic acid conversion rate can be achieved, significantly increasing the yield of the product nicotinamide. Therefore, the glutamine transaminase mutant of this invention has good prospects for industrial application in nicotinamide synthesis.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and more specifically, to a glutamine transaminase mutant and its applications. Background Technology

[0002] Nicotinamide, also known as nicotinamide or vitamin B3, is an amide derivative of nicotinic acid (pyridine-3-carboxylic acid) and belongs to the water-soluble B vitamins. Nicotinamide participates in various physiological processes in the body, including redox reactions, energy metabolism, and DNA repair.

[0003] In the pharmaceutical field, niacinamide is used as an adjunct therapy to treat arrhythmias, myocardial ischemia, and certain skin diseases. In the cosmetics industry, niacinamide, due to its whitening, antioxidant, skin barrier repair, and oil-controlling effects, has become one of the core active ingredients in skincare products. Therefore, the global market demand for niacinamide continues to grow, and the production technology of high-quality niacinamide is attracting significant attention.

[0004] Currently, the industrial production of nicotinamide mainly relies on chemical synthesis. For example, nicotinic acid is reacted with ammonia or ammonia water under high temperature (150-250℃) and high pressure (0.5-2.0 MPa) conditions to produce nicotinamide. While this method is mature and has high yield, it has the following significant drawbacks: harsh reaction conditions, high energy consumption, and large equipment investment; byproducts such as nicotinic acid nitrile and ammonium nicotinate may be generated during the reaction, affecting product quality and requiring multi-step purification; the use of corrosive raw materials such as ammonia poses safety risks, and the wastewater treatment process is demanding, failing to meet the requirements of green chemistry and sustainable development.

[0005] Therefore, developing a new green synthesis method for nicotinamide that features mild reaction conditions, environmental friendliness, high selectivity, and low cost has significant industrial application value and market prospects.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide a glutamine transaminase mutant and its application, which can efficiently catalyze the synthesis of nicotinamide from nicotinic acid.

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a glutamine transaminase mutant, which is obtained by mutation based on the wild-type glutamine transaminase shown in SEQ ID NO.1; the mutation mode is: leucine at position 267 is mutated to glycine, i.e., L267G.

[0009] Among them, wild-type glutamine transaminase (NSPN) is derived from Streptomyces (Streptomyces cerevisiae).Streptomyces murayamaensis During the research and development process, the inventors discovered that this enzyme could convert 3-amino-4-hydroxybenzoic acid to 3-amino-4-hydroxybenzoamide in the biosynthetic pathway of 4-hydroxy-3-nitrobenzamide, but it could not convert nicotinic acid to nicotinamide. To synthesize nicotinamide more efficiently and environmentally, the inventors modified the substrate spectrum of the aforementioned wild-type transglutaminase, enabling it to catalyze the synthesis of nicotinamide from nicotinic acid.

[0010] In some embodiments, the nucleotide sequence encoding wild-type glutamine transaminase is shown in SEQ ID NO.3.

[0011] Furthermore, the present invention provides a glutamine transaminase mutant, which is obtained by mutation based on the wild-type glutamine transaminase shown in SEQ ID NO.1; the mutation mode is: leucine at position 267 is mutated to glycine, and proline at position 315 is mutated to glycine, i.e., L267G / P315G.

[0012] In some embodiments, the above-mentioned glutamine transaminase mutant is mutated as follows: leucine at position 267 is mutated to glycine, and aspartic acid at position 385 is mutated to asparagine, i.e., L267G / D385N.

[0013] In some embodiments, the above-mentioned glutamine transaminase mutant is mutated as follows: leucine at position 267 is mutated to glycine, and proline at position 541 is mutated to serine, i.e., L267G / P541S.

[0014] In some embodiments, the above-mentioned glutamine transaminase mutant is mutated as follows: leucine at position 267 is mutated to glycine, and arginine at position 521 is mutated to glutamic acid, i.e., L267G / R521E.

[0015] Preferably, the mutation mode of the above-mentioned glutamine transaminase mutant is as follows: leucine at position 267 is mutated to glycine, proline at position 315 is mutated to glycine, and aspartic acid at position 385 is mutated to asparagine, i.e., L267G / P315G / D385N.

[0016] More preferably, the mutation mode of the above-mentioned glutamine transaminase mutant is: leucine at position 267 is mutated to glycine, proline at position 541 is mutated to serine, and arginine at position 521 is mutated to glutamic acid, namely L267G / P541S / R521E.

[0017] Most preferably, the mutation mode of the above-mentioned glutamine transaminase mutant is as follows: leucine at position 267 is mutated to glycine, proline at position 315 is mutated to glycine, aspartic acid at position 385 is mutated to asparagine, proline at position 541 is mutated to serine, and arginine at position 521 is mutated to glutamic acid, namely L267G / P315G / D385N / P541S / R521E, and its amino acid sequence is shown in SEQ ID NO.2.

[0018] Secondly, the present invention provides a nucleic acid molecule that encodes the above-mentioned glutamine transaminase mutant.

[0019] In some embodiments, the nucleotide sequence of the glutamine transaminase mutant shown in SEQ ID NO.2 is as shown in SEQ ID NO.4. This nucleotide sequence is obtained by mutating the codon encoding leucine at position 267 to a codon encoding glycine, the codon encoding proline at position 315 to a codon encoding glycine, the codon encoding aspartic acid at position 385 to a codon encoding asparagine, the codon encoding proline at position 541 to a codon encoding serine, and the codon encoding arginine at position 521 to a codon encoding glutamate.

[0020] Thirdly, the present invention provides a recombinant expression vector containing the aforementioned nucleic acid molecules.

[0021] Fourthly, the present invention provides a recombinant bacterium containing the above-mentioned nucleic acid molecules or recombinant expression vectors, or expressing the above-mentioned glutamine transaminase mutant.

[0022] In some embodiments, the starting strain of the recombinant bacteria is *Escherichia coli*. In addition, it can be other commonly used model strains in the art; the present invention does not limit the starting strain.

[0023] The method for constructing the recombinant bacteria can adopt conventional operating procedures in the field. The vector used can be selected according to the chosen starting strain or actual experimental conditions, and the present invention does not limit it.

[0024] In some embodiments, the method for constructing the recombinant bacteria includes: linking the nucleic acid molecules to an expression vector, and then introducing the recombinant expression vector into the starting strain.

[0025] Fifthly, the present invention provides a method for preparing the above-mentioned glutamine transaminase mutant, comprising: inoculating the above-mentioned recombinant bacteria into a fermentation medium and culturing them to obtain the glutamine transaminase mutant.

[0026] In some embodiments, the above-mentioned method for preparing the glutamine transaminase mutant is as follows: using the nucleotide sequence shown in SEQ ID NO:3 as a template, the corresponding mutation is introduced by overlapping extension PCR using site-directed mutagenesis primers, the mutant product is transformed into the starting strain, and the glutamine transaminase mutant expression strain is screened to obtain the strain that is correctly verified by sequencing is the target strain, and the target strain is induced to express the glutamine transaminase mutant.

[0027] Sixthly, the present invention provides the application of the above-mentioned glutamine transaminase mutant, nucleic acid molecule, recombinant expression vector or recombinant bacteria in any of the following: (1) Synthesize nicotinamide using nicotinic acid as a substrate, or indirectly synthesize derivatives of nicotinamide; (2) Prepare products of nicotinamide and its derivatives synthesized from nicotinic acid as substrate.

[0028] The aforementioned nicotinamide derivatives include: β-nicotinamide mononucleotide (NMN), nicotinamide nucleoside (NR), reduced nicotinamide nucleoside (NRH), nicotinamide adenine dinucleotide (NADH), nicotinamide riboside chloride, dihydronicotinamide ribose, thio-oxidized nicotinamide adenine dinucleotide, 6-methylnicotinamide, 1-benzyl-1,4-dihydronicotinamide (BNAH), etc. These nicotinamide derivatives can be prepared from nicotinamide using conventional methods. For example, the preparation of 6-methylnicotinamide involves first obtaining nicotinamide using the method of this invention, and then introducing a methyl group at the 6-position of the nicotinamide pyridine ring using conventional chemical or biosynthetic methods to finally obtain 6-methylnicotinamide.

[0029] In a seventh aspect, the present invention can also provide a biocatalyst comprising the above-mentioned glutamine transaminase mutant or recombinant bacteria. Using this biocatalyst, the substrate nicotinic acid can be converted into nicotinamide.

[0030] Eighthly, the present invention may also provide a method for synthesizing nicotinamide, which uses nicotinic acid as a substrate and adds the above-mentioned glutamine transaminase mutant or recombinant bacteria to the reaction system to catalyze the reaction system to generate nicotinamide.

[0031] In some embodiments, the reaction system includes nicotinic acid, ATP, ammonium source, magnesium ions, phosphate buffer, and the above-mentioned glutamine transaminase mutant or wet bacterial cells of the glutamine transaminase mutant.

[0032] In some embodiments, the reaction system contains: 1-10 mM nicotinic acid, 1-10 mM ATP, 100-600 mM glutamine, 1-10 mM MgCl2, and 2-10 g / L glutamine transaminase mutant or wet cells.

[0033] More preferably, polyphosphate kinase (PPK) is also added to the above reaction system. By combining PPK with the glutamine transaminase mutant of the present invention, the reaction efficiency can be further improved and the cost of ATP usage can be reduced.

[0034] Accordingly, the reaction system contains: 2-20 g / L nicotinic acid, 1-10 mM ATP, 100-400 mM glutamine, 1-10 mM MgCl2, 2-30 mM sodium hexametaphosphate, 2-10 g / L glutamine transaminase mutant or wet cells, and 2-5 g / L polyphosphate kinase.

[0035] In some embodiments, the reaction conditions are: pH = 7~10, temperature = 20~50°C. More preferably, the reaction conditions are: pH = 9, temperature = 30°C.

[0036] The present invention has the following beneficial effects: This invention modifies the substrate profile of streptavidin-derived transglutaminase through protein engineering, resulting in a transglutaminase mutant capable of catalyzing the synthesis of nicotinic acid from nicotinamide. Combined with the synthesis method of this invention, an extremely high nicotinic acid conversion rate is achieved, significantly increasing the yield of nicotinamide. Therefore, the transglutaminase mutant of this invention has promising industrial application prospects in nicotinamide synthesis. Attached Figure Description

[0037] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The process curves for the production of nicotinamide from nicotinic acid using wild-type and penta-mutant catalysis in Example 2 are shown. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0040] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0041] The nicotinic acid and nicotinamide standards used in the examples were purchased from Sigma-Aldrich. The wild-type NSPN gene was derived from Streptomyces (…). Streptomyces murayamaensis The genome sequence was synthesized, and its nucleotide sequence is shown in SEQ ID NO: 3. The corresponding amino acid sequence is as follows: (SEQ ID NO:1).

[0042] Example 1 This example demonstrates the design and gene construction of a glutamine transaminase mutant. The specific steps are as follows: (1) The nucleotide sequence encoding wild-type NSPN (SEQ ID NO: 3) was cloned into plasmid pET-28a(+) to obtain recombinant plasmid pET-28a-NSPN-WT, which was used as a template for subsequent site-directed mutagenesis.

[0043] (2) Based on the homology modeling of NSPN, structural analysis was performed. In order to expand the enzyme substrate spectrum, the key aspartic acid binding pocket was enlarged, and the key loop region for substrate binding and release was flexibly enhanced. Related mutants were designed and site-directed mutagenesis primers were synthesized (Table 1).

[0044] (3) Using pET-28a-NSPN-WT plasmid as a template, PfuUltra TM Overlap extension PCR was performed using high-fidelity DNA polymerase (Stratagene).

[0045] The PCR cycling conditions were: denaturation at 98℃ for 30 s, annealing at 58℃ for 15 s, extension at 72℃ for 4 min, for a total of 33 cycles.

[0046] (4) The amplified product was digested with Dpn I enzyme at 37°C for 1 h to digest the template plasmid, and then transformed into... E.coli DH5α competent cells were plated on LB agar plates containing 50 μg / mL kanamycin. Single clones were picked for sequencing verification.

[0047] (5) Using a step-by-step stacking approach, mutant plasmids containing different mutation combinations were constructed. First, using pET-28a-NSPN-WT as a template, the L267G mutation was introduced to construct pET-28a-NSPN-L267G. Then, using this plasmid as a template, double, triple, and pentamutations containing P315G, D385N, P541S, and R521E were constructed, and the following mutant plasmids were finally obtained: pET-28a-NSPN-L267G; pET-28a-NSPN-L267G / P315G; pET-28a-NSPN-L267G / D385N; pET-28a-NSPN-L267G / P541S; pET-28a-NSPN-L267G / R521E; pET-28a-NSPN-L267G / P315G / D385N; pET-28a-NSPN-L267G / P541S / R521E.

[0048] pET-28a-NSPN-L267G / P315G / D385N / P541S / R521E (The nucleotide sequence of the gene encoded by this mutant is shown in SEQ ID NO:4, and the amino acid sequence is shown in SEQ ID NO:2).

[0049] The mutant primers are shown in Table 1: Table 1. Site-directed mutagenesis primer sequences

[0050] Example 2 This example demonstrates the construction and catalytic activity screening of mutant strains. 1. Construction of mutant strains: Each mutant plasmid correctly constructed in Example 1 was transformed into the expression host. E.coli BL21(DE3) competent cells were plated on LB agar plates containing 50 μg / mL kanamycin. Single colonies were picked, cultured, and sequenced for verification. Strains with correct sequencing results were retained. The final mutant strain was constructed as follows: pET-28a-NSPN-L267G; pET-28a-NSPN-L267G / P315G; pET-28a-NSPN-L267G / D385N; pET-28a-NSPN-L267G / P541S; pET-28a-NSPN-L267G / R521E; pET-28a-NSPN-L267G / P315G / D385N; pET-28a-NSPN-L267G / P541S / R521E; pET-28a-NSPN-L267G / P315G / D385N / P541S / R521E.

[0051] Strains containing the pET-28a-NSPN-WT plasmid were used as controls.

[0052] 2. Preparation of crude enzyme solution and determination of catalytic ability: Single colonies of each strain were inoculated into 5 mL of LB liquid medium (containing 50 μg / mL kanamycin) and cultured overnight at 37°C with shaking at 220 rpm. A 1% inoculum was then transferred to 50 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with 220 rpm until OD600 ≈ 0.8. IPTG was added to a final concentration of 0.5 mM, and expression was induced at 25°C for 18 h. Wet cells were collected by centrifugation at 6000 rpm for 10 min at 4°C.

[0053] Take an appropriate amount of wet bacterial cells and resuspend them in 50 mM, pH 7.0 potassium phosphate buffer to a bacterial concentration of 40 g / L. Place the resuspended solution in an ice-water bath and sonicate for 10 min, then centrifuge at 4℃ and 12000 rpm for 10 min. Collect the supernatant as crude enzyme solution for later use.

[0054] The catalytic reaction system (1 mL) consisted of: crude enzyme solution (equivalent to 10 g / L bacterial cells), nicotinic acid 5 mM, ATP 10 mM, glutamine 100 mM, MgCl2 10 mM, and 50 mM, pH 9.0 potassium phosphate buffer was added to make up the volume. The reaction was carried out at 37℃ and 200 rpm for 5 h. After the reaction was completed, 100 μL of 1 M trichloroacetic acid was added to terminate the reaction, and the mixture was centrifuged at 12000 rpm for 10 min. The supernatant was appropriately diluted and the amount of nicotinamide produced was analyzed by HPLC. Three replicate experiments were conducted for each strain.

[0055] The yield and conversion rate of nicotinamide from nicotinic acid catalyzed by crude enzyme solution of each strain over 12 h are shown in Table 2. Figure 1 Process curves for the production of nicotinamide from nicotinic acid using NSPN catalysis for wild-type and penta-mutant strains.

[0056] Table 2. Nicotinamide yield of each strain after 12 h of catalysis with crude enzyme solution

[0057] The results showed that wild-type NSPN had no catalytic activity for nicotinic acid, while all mutants constructed in this invention acquired the ability to catalyze the conversion of nicotinic acid to nicotinamide. Among them, the genetically engineered bacteria containing the five mutants (L267G / P315G / D385N / P541S / R521E) exhibited the highest catalytic activity, with a conversion rate of 96% after 12 h, demonstrating good prospects for industrial application.

[0058] Example 3 This example demonstrates the purification of the five mutants and the characterization of the pure enzyme's enzymatic properties. To further investigate the catalytic properties of the optimal glutamine transaminase mutant (L267G / P315G / D385N / P541S / R521E, hereinafter referred to as NSPN-M5, whose amino acid sequence is shown in SEQ ID NO:2), this embodiment isolates and purifies NSPN-M5 to obtain high-purity enzyme protein, and characterizes its basic enzymatic properties.

[0059] 1. Induction and purification of recombinant NSPN-M5 The genetically engineered bacteria containing the pET-28a-NSPN-L267G / P315G / D385N / P541S / R521E plasmid constructed in Example 2 were used. E. coliBL21(DE3) cells were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured overnight at 37°C with shaking at 220 rpm. A 1% inoculum was then transferred to 1 L of LB medium (containing 50 μg / mL kanamycin) and cultured under the same conditions until OD600 ≈ 0.8. IPTG was added to a final concentration of 0.1 mM, and expression was induced at 18°C ​​for 18 h. Wet cells were collected by centrifugation at 6000 rpm for 15 min at 4°C.

[0060] Take 10 g of the above wet bacterial cells and add buffer A (50 mM potassium phosphate buffer, pH 8.0, containing 300 mM NaCl and 10 mM imidazole) at a ratio of 1:10 (g / mL), and resuspend evenly. Hypolyze the bacterial cells three times using a high-pressure homogenizer at 4℃ and 800 bar. Centrifuge the lysate at 4℃ and 12000 rpm for 30 min, collect the supernatant, and filter through a 0.45 μm filter membrane to obtain a cell-free crude extract.

[0061] Cell-free crude extract was loaded onto a Ni-NTA affinity chromatography column (5 mL) pre-equilibrated with buffer A at a flow rate of 1 mL / min. Impurities were eluted sequentially with 10 column volumes of buffer A and 10 column volumes of buffer B (50 mM potassium phosphate buffer, pH 8.0, containing 300 mM NaCl and 50 mM imidazole). Finally, the target protein was eluted with buffer C (50 mM potassium phosphate buffer, pH 8.0, containing 300 mM NaCl and 250 mM imidazole), and the elution peak was collected. The eluent was concentrated and transferred to storage buffer (50 mM potassium phosphate buffer, pH 8.0, containing 10% glycerol). Protein concentration was determined using the Bradford method, and protein purity was assessed by SDS-PAGE electrophoresis.

[0062] The results showed that the purified NSPN-M5 appeared as a single band on the SDS-PAGE gel, with a molecular weight of approximately 69 kDa, consistent with the theoretical molecular weight.

[0063] 2. Optimal reaction pH of pure enzymes Take an appropriate amount of purified NSPN-M5 and dilute it to the same concentration with 50 mM buffers of different pH values ​​(pH 6.0-10.0) (potassium phosphate buffer for pH 6.0-7.0, Tris-HCl buffer for pH 8.0-9.0, and glycine-sodium hydroxide buffer for pH 10.0-11.0).

[0064] The catalytic reaction system described in Example 2 (nicotinic acid 5 mM, ATP 10 mM, glutamine 100 mM, MgCl2 10 mM, final enzyme concentration 0.5 mg / mL) was reacted at 37°C for 120 min, and the initial reaction rate was measured under various pH conditions. The optimal reaction pH was determined by calculating the relative enzyme activity, with the highest enzyme activity defined as 100%.

[0065] The measurement results are shown in Table 3: Table 3 Enzyme activities at different pH levels

[0066] The results in Table 3 show that NSPN-M5 exhibits the best enzyme activity at pH 9.0.

[0067] 3. Optimal reaction temperature of pure enzymes At the optimal reaction pH (pH 9.0), the reaction system was placed in a water bath at 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. The initial reaction rate was determined according to the method described above, and the optimal reaction temperature was determined.

[0068] The measurement results are shown in Table 4: Table 4 Enzyme activities at different temperatures

[0069] The results showed that the relative activities of NSPN-M5 were 94%, 100%, 98%, 78%, 41%, and 22% at temperatures of 25℃, 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. The results indicated that the enzyme activity of NSPN-M5 was optimal at 30℃.

[0070] Example 4 This example demonstrates the cascade catalytic production of nicotinamide using the mutant NSPN-M5 and its condition optimization. To further improve reaction efficiency and reduce ATP usage costs, this embodiment introduces polyphosphate kinase (PPK) into the reaction system to achieve in-situ ATP regeneration.

[0071] Crude enzyme solutions for each mutant were prepared using the same method as in Example 2. The glutamine concentration in this system was optimized. The one-pot cascade reaction system (100 mL) is as follows: Cell lysis buffer (equivalent to 10 g / L bacterial cells), nicotinic acid 20 g / L, ATP 10 mM, glutamine 100 mM, 200 mM, 400 mM, 600 mM and 800 mM, MgCl2 20 mM, polyphosphoric acid kinase (RsPPK, InvivoChem) 5 g / L, sodium hexametaphosphate 30 mM, and replenished to volume with 100 mM, pH 9.0 potassium phosphate buffer.

[0072] After the reaction system was prepared, it was reacted in a shaker at 30℃ and 200 rpm for 12 h. Samples were taken at the beginning of the reaction (immediately after the addition of the substrate nicotinic acid) and after the reaction was completed. The reaction was terminated by adding 1 M trichloroacetic acid. After centrifugation, the supernatant was taken for HPLC analysis to determine the amount of nicotinamide produced.

[0073] The reaction results are shown in Table 5.

[0074] Table 5. Nicotinamide yield of different mutants in the cascaded PPK system after 12 h

[0075] Among them, NSPN-M5 showed the highest conversion rate at a 200 mM amino donor concentration, almost completely converting 20 g / L nicotinic acid within 12 h, with a nicotinamide yield of 19.62 g / L and a conversion rate as high as 98.1%. This result further confirms the great potential of the glutamine transaminase mutant described in this invention in the biosynthesis of nicotinamide.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A glutamine transaminase mutant, characterized by, The glutamine transaminase mutant was obtained by mutation based on the wild-type glutamine transaminase shown in SEQ ID NO. 1; The mutation mode of the mutant includes: mutating leucine at position 267 to glycine.

2. The transglutaminase mutant according to claim 1, characterized by The glutamine transaminase mutant was obtained by mutation based on the wild-type glutamine transaminase shown in SEQ ID NO.1; The mutation mode of the mutant is: leucine at position 267 is mutated to glycine, and proline at position 315 is mutated to glycine; or The mutation mode of the mutant is as follows: leucine at position 267 is mutated to glycine, and aspartic acid at position 385 is mutated to asparagine; or The mutation mode of the mutant is as follows: leucine at position 267 is mutated to glycine, and proline at position 541 is mutated to serine; or The mutation mode of the mutant is as follows: leucine at position 267 is mutated to glycine, and arginine at position 521 is mutated to glutamic acid; or The mutation mode of the mutant is as follows: leucine at position 267 is mutated to glycine, proline at position 315 is mutated to glycine, and aspartic acid at position 385 is mutated to asparagine; or The mutation mode of the mutant is as follows: leucine at position 267 is mutated to glycine, proline at position 541 is mutated to serine, and arginine at position 521 is mutated to glutamic acid; or The mutant is mutated as follows: leucine at position 267 is mutated to glycine, proline at position 315 is mutated to glycine, aspartic acid at position 385 is mutated to asparagine, proline at position 541 is mutated to serine, and arginine at position 521 is mutated to glutamic acid.

3. A nucleic acid molecule, characterized in that, Encodes the glutamine transaminase mutant of claim 1 or 2.

4. A recombinant expression vector, characterized in that, It contains the nucleic acid molecule as described in claim 3.

5. A recombinant bacterium, characterized in that, It contains the nucleic acid molecule of claim 3 or the recombinant expression vector of claim 4, or expresses the glutamine transaminase mutant of claim 1 or 2.

6. A method for preparing the glutamine transaminase mutant according to claim 1 or 2, characterized in that, include: The recombinant bacteria of claim 5 were inoculated into a fermentation medium and cultured to obtain the glutamine transaminase mutant.

7. The use of the glutamine transaminase mutant of claim 1 or 2, the nucleic acid molecule of claim 3, the recombinant expression vector of claim 4, or the recombinant bacteria of claim 5 in any of the following: (1) Synthesis of nicotinamide and its derivatives using nicotinic acid as a substrate; (2) Prepare products of nicotinamide and its derivatives synthesized from nicotinic acid as substrate.

8. A biocatalyst, characterized in that, Includes the glutamine transaminase mutant of claim 1 or 2 or the recombinant bacteria of claim 5; Preferably, the biocatalyst further includes polyphosphokinase.

9. A method for synthesizing nicotinamide, characterized in that, Using nicotinic acid as a substrate, the glutamine transaminase mutant as described in claim 1 or 2 or the recombinant bacteria as described in claim 5 is added to the reaction system to catalyze the generation of nicotinamide.

10. The method according to claim 9, characterized in that, The reaction system contains nicotinic acid, ATP, ammonium source, magnesium ions, phosphate buffer, and the glutamine transaminase mutant of claim 1 or 2 or wet bacterial cells expressing the glutamine transaminase mutant of claim 1 or 2. Preferably, the reaction system contains: 1-10 mM nicotinic acid, 1-10 mM ATP, 100-600 mM glutamine, 1-10 mM MgCl2, and 2-10 g / L glutamine transaminase mutant or wet bacterial cells; Preferably, the reaction system contains: 2-20 g / L nicotinic acid, 1-10 mM ATP, 100-400 mM glutamine, 1-10 mM MgCl2, 2-30 mM sodium hexametaphosphate, 2-10 g / L glutamine transaminase mutant or wet bacterial cells, and 1-5 g / L polyphosphate kinase; Preferably, the reaction conditions are: pH = 7~10, temperature = 20~40℃; Preferably, the reaction conditions are: pH=9 and temperature=30℃.

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