An imine reductase mutant and its application in synthesis of 3-methyl-10-ethyl deazaflavin

Abstract

The present application relates to an imine reductase mutant and its application in synthesis of 2-methyl-10-ethyl deazaflavin. The amino acid sequence of the imine reductase mutant is shown in SEQ ID NO. 2, and the nucleotide sequence is shown in SEQ ID NO. 1. The present application also provides a method for synthesizing 2-methyl-10-ethyl deazaflavin by using the imine reductase mutant, which comprises the following steps: (1) preparing supernatant containing the imine reductase mutant by a heterologous expression method; (2) preparing solution A and B respectively by using (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione, D-glucose, glutamate dehydrogenase and reduced nicotinamide adenine dinucleotide phosphate as raw materials; and (3) reacting solution A and B to prepare 2-methyl-10-ethyl deazaflavin. The imine reductase mutant and the synthesis method provided by the present application overcome the shortcomings of chemical preparation method, such as complex route, high cost, difficult purification, serious environmental pollution and the like, and provide a green approach for synthesis of 2-methyl-10-ethyl deazaflavin.

Description

Technical Field

[0001] This invention relates to an imine reductase mutant and its application in the synthesis of 3-methyl-10-ethyl desoxyflavin, belonging to the field of genetic engineering technology. Background Technology

[0002] As we age, the amount of collagen in our skin gradually decreases, and the structure and function of collagen fibers also change, leading to loss of skin elasticity and the appearance of wrinkles. Therefore, supplementing collagen and promoting collagen synthesis are important ways to combat wrinkles.

[0003] Denitroflavins are an important class of bioactive molecules that participate in various biological redox reactions as coenzyme analogs, and have wide applications in medicine, agriculture, and biocatalysis. 3-Methyl-10-ethyldenitroflavin, as a derivative of denitroflavin, exhibits high biological activity and can promote NAD+ metabolism in vivo. + It is produced and can activate longevity proteins sirtuins, which helps delay aging and can also repair mitochondrial membrane potential and improve ATP production efficiency; it has good stability and can maintain its activity and effect under different environmental and storage conditions; it has stronger antioxidant properties and can neutralize free radicals generated during mitochondrial electricity production, creating a stable internal environment for mitochondria; it can directly function as a coenzyme and can directly insert into the "energy pore" of mitochondria to activate links related to energy metabolism.

[0004] Traditional methods for synthesizing denitroflavin compounds largely rely on pure chemical synthesis, which suffers from cumbersome reaction steps, poor stereoselectivity, the need for toxic reagents (such as heavy metal reducing agents), and low environmental friendliness. In recent years, biocatalysis technology, due to its advantages of mild conditions, high selectivity, and environmental friendliness, has been increasingly applied to the synthesis of complex compounds. *Aspergillus niger*, a widely distributed filamentous fungus, encodes several imine reductases with potential catalytic activity in its genome. These enzymes can catalyze the reductive coupling reactions of various carbonyl compounds with amines. When both the carbonyl compound and the amine exhibit high reactivity, a 1:1 substrate ratio can be used for the reaction, providing a green route for the synthesis of nitrogen-containing heterocyclic compounds.

[0005] However, there are currently no reports on the enzymatic synthesis of 3-methyl-10-ethyldeoxyflavin. Therefore, it is of great significance to develop an imine reductase that can synthesize 3-methyl-10-ethyldeoxyflavin, as well as an efficient synthetic route that combines chemical and enzymatic methods. Summary of the Invention

[0006] To address the shortcomings of existing technologies, an imine reductase mutant and its application in the synthesis of 3-methyl-10-ethyl desoxyflavin are proposed.

[0007] The technical solution of the present invention is as follows: An imine reductase mutant, the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene of which is shown in SEQ ID NO.1.

[0008] The imine reductase mutant provided by this invention is derived from Aspergillus niger ( Aspergillus niger Based on the imine reductase gene, the following mutations were made: valine at position 106 was mutated to aspartic acid (Asp); methionine at position 214 was mutated to isoleucine (Ile); aspartic acid at position 226 was mutated to tyrosine (Tyr); glutamine at position 233 was mutated to proline (Pro); and alanine at position 266 was mutated to glutamate (Glu). The GenBank accession number for the imine reductase is KY327363.1.

[0009] One recombinant vector involves inserting the encoding gene of the aforementioned imine reductase mutant into a plasmid vector.

[0010] According to a preferred embodiment of the present invention, the plasmid vector is pET-28a(+).

[0011] A recombinant strain is obtained by transforming the above-mentioned recombinant vector into a host cell.

[0012] According to a preferred embodiment of the present invention, the host cell is Escherichia coli.

[0013] Application of the above-mentioned imine reductase mutant in the synthesis of 3-methyl-10-ethyl desoxyflavin.

[0014] A method for synthesizing 3-methyl-10-ethyl desoxyflavin includes the following steps: (1) The imine reductase mutant gene was cloned into an expression vector to obtain a recombinant expression vector; then the recombinant expression vector was transformed into host cells, positive clones were selected for fermentation culture, and wet cells were collected; then the obtained wet cells were added to PBS buffer, sonicated for 35-45 min, and then centrifuged at 8000-12000 rpm for 5-15 min to obtain a supernatant containing the imine reductase mutant; (2) Dissolve (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in dimethyl sulfoxide (DMSO) and stir until completely dissolved to obtain solution A; add D-glucose, glutamate dehydrogenase (GDH), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) to PBS buffer to obtain solution B; (3) Add solution A and the supernatant containing the imine reductase mutant obtained in step (1) to solution B, stir and mix evenly, react at 35~40℃ and 200~300rpm for 20~30h, then add NaOH to terminate the reaction, and after extraction and drying, 3-methyl-10-ethyl denitrifying flavin is obtained.

[0015] According to a preferred embodiment of the present invention, in step (1), the PBS buffer pH=7.5, the mass-to-volume ratio of the PBS buffer to the wet bacterial cells is 10:1, unit: mL / g; the ultrasonic disruption is performed using an ultrasonic cell disruptor at a power of 150~300kw for 40min, with a working time of 3s and a stop time of 3s.

[0016] According to a preferred embodiment of the present invention, in step (2), the structural formula of the (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione is shown in formula (1) below: ; The preparation is carried out according to the following method: 1-Methylbarbituric acid and 2-ethylaminobenzaldehyde were added to a reaction vessel containing ethanol to construct a reaction system. The reaction system was stirred at 60-90℃ for 4-8 h. After the reaction was completed, the reaction solution was cooled to room temperature, a solid precipitated, filtered, and recrystallized with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione. The mass ratio of 1-methylbarbituric acid to 2-ethylaminobenzaldehyde is 1:(1~1.2); the volume mass ratio of ethanol to 1-methylbarbituric acid is (10~20):1, unit: mL / g.

[0017] According to a preferred embodiment of the present invention, in step (2), the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in solution A is 0.2~0.3 g / mL.

[0018] According to a preferred embodiment of the present invention, in step (2), the concentration of D-glucose in solution B is 55-65 mM, the concentration of glutamate dehydrogenase (GDH) is 55-65 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) is 35-45 mM.

[0019] According to a preferred embodiment of the present invention, in step (3), the volume ratio of solution A, solution B and supernatant containing imine reductase mutant is (45~55):(440~460):10.

[0020] According to a preferred embodiment of the present invention, in step (3), the termination reaction, extraction, and drying specifically refer to: First, 5M NaOH was added to terminate the reaction. Then, the mixture was extracted twice with 3 times the volume of ethyl acetate. The extracted organic phases were combined and finally dried with anhydrous MgSO4.

[0021] Technical features and beneficial effects of the present invention: 1. This invention is based on imine reductase derived from Aspergillus niger (GenBank accession number: KY327363), and finally screened to obtain imine reductase mutants with the following mutations: valine at position 106 mutated to aspartic acid (Asp), methionine at position 214 mutated to isoleucine (Ile), aspartic acid at position 226 mutated to tyrosine (Tyr), glutamine at position 233 mutated to proline (Pro), and alanine at position 266 mutated to glutamate (Glu). These mutants are then used in the synthesis of 3-methyl-10-ethyldeoxyflavin.

[0022] 2. Compared with wild-type imine reductase, the imine reductase mutant provided by this invention has significantly enhanced enzyme activity, and can more effectively catalyze the reductive coupling reaction between (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione and D-glucose, thereby improving the utilization rate of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione and greatly improving the synthesis efficiency and purity of 3-methyl-10-ethyl desoxyflavin.

[0023] 3. The method for synthesizing 3-methyl-10-ethyldeazoflavin provided by this invention overcomes the disadvantages of complex chemical preparation routes, high costs, difficult purification, and serious environmental pollution, providing a green route for the synthesis of 3-methyl-10-ethyldeazoflavin. Furthermore, the reaction steps are short and the conditions are mild, significantly reducing equipment investment and energy costs, making it suitable for industrial-scale production. Attached Figure Description

[0024] Figure 1 Agarose gel electrophoresis images of imine reductase mutant V106D-M214I-D226Y-Q233P-A266E and wild-type imine reductase; In the figure, lane 1 represents wild-type imine reductase, and lane 2 represents imine reductase mutant.

[0025] Figure 2 This is a liquid phase diagram of 3-methyl-10-ethyldeoxyflavin prepared in Example 4.

[0026] Figure 3 The liquid phase diagram is for 3-methyl-10-ethyldeoxyflavin prepared in Comparative Example 1. Detailed Implementation

[0027] The following embodiments and accompanying drawings are merely illustrative of specific implementation schemes for carrying out the present invention. These schemes and drawings should not be construed as limiting the present invention. Any changes made without departing from the principles and essence of the present invention shall fall within the protection scope of the present invention.

[0028] Unless otherwise specified, the experimental techniques and methods used in this embodiment are conventional. Unless otherwise specified, all materials and reagents used in this embodiment can be obtained through legitimate commercial channels.

[0029] To better explain this invention, the following embodiments further illustrate the invention. Unless otherwise specified, the experimental methods in this invention are conventional methods. For specific molecular biology experimental methods not specifically described, please refer to *Molecular Cloning: A Laboratory Manual* edited by J. Sambrook et al., or follow the kit instructions. Unless otherwise specified, the reagents and biological materials used in the specific embodiments are commercially available.

[0030] Example 1: Selection of mutation sites Homology modeling was performed on the amino acid sequence shown in the wild-type imine reductase nucleotide sequence (GenBank accession number: KY327363.1), and then docked with the substrate molecule. The results were then imported into Discovery Studio software for visualization analysis. The amino acids that interact with the substrate were analyzed, and V106, M214, D226, Q233, and A266 were selected as potential mutation sites.

[0031] Example 2: Preparation of imine reductase mutant 1. The nucleotide sequence of wild-type imine reductase (GenBank accession number: KY327363.1) was artificially synthesized by Genewiz Biotechnology Co., Ltd., and its coding gene sequence was then cloned into the pET-28a plasmid to obtain the recombinant plasmid pET-28a-AspRedAm expressing wild-type imine reductase AspRedAm. The recombinant plasmid was then transformed into Escherichia coli BL21 (DE3) competent cells by calcium chloride method to obtain recombinant Escherichia coli BL21-pET-28a-AspRedAm expressing imine reductase AspRedAm, and stored at -80℃.

[0032] 2. Through five rounds of iterative saturation mutation and preliminary screening of dominant strains, the AspRedAm mutant gene was obtained. The specific methods are as follows: Round 1: Using the recombinant plasmid pET-28a-AspRedAm as a template and F106 and R106 as primers, site-directed saturation mutagenesis PCR was performed to mutate the valine at position 106 of its amino acid sequence to the remaining 19 amino acids. The resulting product was then transformed into E. coli, plated on LB solid medium, and cultured. Through screening of dominant strains, recombinant E. coli BL21-pET-28a-AspRedAm-V106D and 18 other recombinant E. coli strains were obtained. Screening method for dominant strains: Select mutant single colonies from LB solid medium and inoculate them into 96-well plates containing 500 μL of LB liquid medium. Incubate at 37℃ and 220 rpm for 12 h. Inoculate 2% (v / v) into 96-well plates containing 500 μL of LB liquid medium and incubate at 37℃ and 220 rpm for 3 h. Add IPTG to a final concentration of 0.1 mmol / L and induce culture at 20℃ for 12 h to obtain fermentation broth. Centrifuge the fermentation broth to obtain wet cells. Sonicate the wet cells to obtain the supernatant containing the imine reductase mutant.

[0033] 14 mg of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6 (1H,3H,5H)-trione was dissolved in 50 µL LDMSO to obtain solution A; Take 450 µL of phosphate buffer (pH 7.5), and add 60 mM D-glucose, 60 mg / mL GDH, 40 mM NADPH, and 10 µL of the supernatant containing the imine reductase mutant sequentially. Stir thoroughly until completely dissolved to obtain solution B. Mix solutions A and B and react in a shaker at 37 °C and 250 rpm for 24 h. Then, add 5 M NaOH to terminate the reaction, extract twice with ethyl acetate, combine the organic phases, and take an appropriate amount for HPLC analysis. The strain with high 3-methyl-10-ethyldeoxyflavin content is the dominant strain, and the screening result is recombinant Escherichia coli BL21-pET-28a-AspRedAm-V106D.

[0034] Round 2: Using the recombinant plasmid extracted from strain BL21-pET-28a-AspRedAm-V106D as a template, and using F214 and R214 as primers, site-directed saturation mutagenesis PCR was performed to mutate methionine at position 214 of the amino acid sequence to the remaining 19 amino acids. The resulting plasmid was then transformed into E. coli, plated on LB solid medium, and cultured. The dominant strain was screened according to the method described in Round 1 to obtain recombinant E. coli BL21-pET-28a-AspRedAm-V106D-M214I.

[0035] Round 3: Using the recombinant plasmid extracted from strain BL21-pET-28a-AspRedAm-V106D-M214I as a template, and with F226 and R226 as primers, saturated mutagenesis PCR was performed to mutate the aspartic acid at position 226 of its amino acid sequence to the remaining 19 amino acids. Then, it was transformed into E. coli, plated on LB solid medium and cultured. The dominant strain was screened according to the method described in Round 1 to obtain recombinant E. coli BL21-pET-28a-AspRedAm-V106D-M214I-D226Y.

[0036] Round 4: Using the recombinant plasmid extracted from strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y as a template, and using F233 and R233 as primers, site-directed saturation mutagenesis PCR was performed to mutate glutamine at position 233 of the amino acid sequence to the remaining 19 amino acids. The resulting plasmid was then transformed into E. coli, plated on LB solid medium, and cultured. The dominant strain was screened according to the method described in Round 1 to obtain recombinant E. coli BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P.

[0037] Round 5: Using the recombinant plasmid extracted from strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P as a template, and with F266 and R266 as primers, saturated mutagenesis PCR was performed to mutate the alanine at position 266 of its amino acid sequence to the remaining 19 amino acids. The resulting plasmid was then transformed into E. coli, plated on LB solid medium, and cultured. The dominant strain was screened according to the method described in Round 1 to obtain recombinant E. coli BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266E.

[0038] The system (25 μL) for the site-directed saturation mutagenesis PCR is shown in Table 1 below.

[0039] Table 1 PCR conditions: 95℃ pre-denaturation for 5 min, followed by 30 cycles: 90℃ for 30 s, 62℃ for 30 s, 72℃ for 7 min, and a final extension at 72℃ for 5 min.

[0040] The primers for the site-directed saturation mutagenesis PCR are shown in Table 2.

[0041] Table 2 Following the above methods, the following 13 recombinant Escherichia coli strains were constructed and preliminarily screened: BL21-pET-28a-AspRedAm-V106D, BL21-pET-28a-AspRedAm-V106E, BL21-pET-28a-AspRedAm-V106D-M214I, BL21-pET-28a-AspRedAm-V106D-M214L, BL21-pET-28a-AspRedAm-V106D-M214V, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y, BL21-pET-28a-AspRedAm-V106D-M214I-D226F ... T-28a-AspRedAm-V106D-M214I-D226W, BL21-pET-28a-AspRedAm-V106D-M214I-D22 6Y-Q233P, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233G, BL21-pET-28a-As pRedAm-V106D-M214I-D226Y-Q233P-A266E, BL21-pET-28a-AspRedAm-V106D-M214I -D226Y-Q233P-A266D, BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266Q.

[0042] 3. Rescreening of enzyme mutants (1) Preparation of enzyme mutants: Taking the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E as an example, strain BL21-pET-28a-AspRedAm-V106D-M214I-D226Y-Q233P-A266E was inoculated into LB liquid medium containing a final concentration of 50 mg / L kanamycin and cultured for 12 h. Then, it was inoculated into fresh cells at an inoculation rate of 2% (v / v). The culture was carried out in LB liquid medium containing a final concentration of 50 mg / L kanamycin until the OD600 value reached 0.6-0.8. Then, IPTG was added to a final concentration of 0.1 mmol / L, and the culture was induced at 20℃ for 12 h to obtain the fermentation broth. The fermentation broth was centrifuged to obtain wet cells. The wet cells were added to phosphate buffer and sonicated, then centrifuged again to obtain the supernatant, which was the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E.

[0043] Wild-type imine reductase, imine reductase mutant V106D, imine reductase mutant V106E, imine reductase mutant V106D-M214I, imine reductase mutant V106D-M214L, imine reductase mutant V106D-M214V, imine reductase mutant V106D-M214I-D226Y, imine reductase mutant V106D-M214I-D226F, imine reductase mutant V106D-M214I-D226W, and imine reductase were prepared using the same method. The mutants V106D-M214I-D226Y-Q233P, V106D-M214I-D226Y-Q233G, V106D-M214I-D226Y-Q233P-A266E, V106D-M214I-D226Y-Q233P-A266D, V106D-M214I-D226Y-Q233P-A266Q, and the wild-type imine reductase AspRedAm were identified.

[0044] DNA agarose gel electrophoresis was performed to verify the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E and the wild-type imine reductase. The results are as follows: Figure 1 As shown.

[0045] Depend on Figure 1 As can be seen, the appearance of the target band in the gel electrophoresis image and the single band indicate that the imine reductase gene mutation was successful. Furthermore, the present invention successfully prepared the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E through heterologous expression.

[0046] (2) The following assays were performed according to the method described in point 2 to determine the levels of wild-type imine reductase, imine reductase mutant V106D, imine reductase mutant V106E, imine reductase mutant V106D-M214I, imine reductase mutant V106D-M214L, imine reductase mutant V106D-M214V, imine reductase mutant V106D-M214I-D226Y, imine reductase mutant V106D-M214I-D226F, imine reductase mutant V106D-M214I-D226W, and imine reductase mutant V1 The catalytic efficiencies of the imine reductase mutants V106D-M214I-D226Y-Q233P, V106D-M214I-D226Y-Q233G, V106D-M214I-D226Y-Q233P-A266E, V106D-M214I-D226Y-Q233P-A266D, and V106D-M214I-D226Y-Q233P-A266Q in the synthesis of 3-methyl-10-ethyldenitrosin are shown in Table 3.

[0047] Table 3 As shown in Table 3, the enzyme activity of the imine reductase mutants was higher than that of the wild-type imine reductase. Among them, the imine reductase mutant V106D-M214I-D226Y-Q233P-A266E showed the best catalytic effect on the synthesis of 3-methyl-10-ethyl desoxyflavin, with a content of 98.5%, which was more than 20% higher than that of the wild-type imine reductase.

[0048] Example 3: Preparation of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione A method for preparing (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione, comprising the following steps: 15 g of 1-methylbarbituric acid and 15 g of 2-ethylaminobenzaldehyde were added to a reaction vessel containing 225 mL of ethanol to construct a reaction system. The reaction system was stirred at 75 °C for 5 h. After the reaction was completed, the reaction solution was cooled to room temperature, a solid precipitated, filtered, and recrystallized with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione.

[0049] Example 4: Synthesis of 3-methyl-10-ethyldezoflavin A method for synthesizing 3-methyl-10-ethyl desoxyflavin includes the following steps: (1) Dissolve 14g of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in 50mL of dimethyl sulfoxide (DMSO) and stir until completely dissolved to obtain solution A; add D-glucose, glutamate dehydrogenase (GDH), and reduced nicotinamide adenine dinucleotide phosphate (NADPH) to 450mL of PBS buffer to obtain solution B; In solution A, the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione was 0.28 g / mL; in solution B, the concentration of D-glucose was 60 mM, the concentration of glutamate dehydrogenase (GDH) was 60 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate (NADPH) was 40 mM. (2) Add 450 mL of solution A and 10 mL of the supernatant containing the imine reductase mutant obtained in Example 2 to 50 mL of solution B, stir and mix evenly, react at 37 °C and 250 rpm for 24 h, then add 5 M NaOH to terminate the reaction, then extract twice with 3 times the volume of ethyl acetate, combine the extracted organic phases, and finally dry the combined organic phases with anhydrous MgSO4 to obtain 3-methyl-10-ethyl dehydroflavin.

[0050] Comparative Example 1 A method for synthesizing 3-methyl-10-ethyl desoxyflavin, the specific steps are the same as in Example 4, except that in step (2), the supernatant containing wild-type imine reductase is used instead of the supernatant containing imine reductase mutant.

[0051] Test case The purity of 3-methyl-10-ethyldeoxyflavin synthesized in Example 4 and Comparative Example 1 was determined by high performance liquid chromatography (HPLC). The specific method is as follows: The Waters Arc 2489 system was used, with an Arlchrom WP C column. 18 4.6*250mm, 5μm; Detector: UV detector; Detection wavelength: 270nm; Mobile phase A: 0.01M potassium dihydrogen phosphate solution, Mobile phase B: acetonitrile; Flow rate: 1mL / min; Column temperature: 15℃; Injection volume: 10uL; Gradient run table is shown below: The high-performance liquid chromatography (HPLC) results of the synthesized 3-methyl-10-ethyl desoxyflavin in Example 4 and Comparative Example 1 are as follows: Figure 2 , Figure 3As shown.

[0052] Depend on Figure 2 , Figure 3 It can be seen that the purity of the 3-methyl-10-ethyldeoxyflavin synthesized in Example 4 reached 98.4%, while the purity of the 3-methyl-10-ethyldeoxyflavin synthesized in Comparative Example 1 was only about 76%. This indicates that compared with wild-type imine reductase, the enzyme activity of the imine reductase mutant provided by the present invention is significantly improved, and it can more effectively catalyze the reductive coupling reaction of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione with D-glucose, thereby improving the utilization rate of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione and greatly improving the synthesis efficiency and purity of 3-methyl-10-ethyldeoxyflavin.

[0053] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. 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. An imine reductase mutant, characterized in that, The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence encoding the gene is shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that, The encoding gene of the imine reductase mutant as described in claim 1 is inserted into a plasmid vector.

3. The recombinant vector as described in claim 2, characterized in that, The plasmid vector is pET-28a(+).

4. A recombinant bacterial strain, characterized in that, It is obtained by converting the recombinant vector described in claim 2 into host cells.

5. The recombinant strain according to claim 4, characterized in that, The host cell is Escherichia coli.

6. The use of the imine reductase mutant of claim 1 in the synthesis of 3-methyl-10-ethyl desoxyflavin.

7. A method for synthesizing 3-methyl-10-ethyldeoxyflavin, characterized in that, The steps include the following: (1) The imine reductase mutant gene of claim 1 is cloned into an expression vector to obtain a recombinant expression vector; then the recombinant expression vector is transformed into a host cell, positive clones are selected for fermentation culture, and wet cells are collected; then the obtained wet cells are added to PBS buffer, sonicated for 35-45 min, and then centrifuged at 8000-12000 rpm for 5-15 min to obtain a supernatant containing the imine reductase mutant; (2) Dissolve (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in dimethyl sulfoxide and stir until completely dissolved to obtain solution A; add D-glucose, glutamate dehydrogenase, and reduced nicotinamide adenine dinucleotide phosphate to PBS buffer to obtain solution B; (3) Add solution A and the supernatant containing the imine reductase mutant obtained in step (1) to solution B, stir and mix evenly, react at 35~40℃ and 200~300rpm for 20~30h, then add NaOH to terminate the reaction, and after extraction and drying, 3-methyl-10-ethyl denitrifying flavin is obtained.

8. The synthesis method according to claim 7, characterized in that, In step (1), the PBS buffer pH=7.5 and the mass-to-volume ratio of the PBS buffer to the wet cells is 10:1, unit: mL / g; the ultrasonic disruption is performed using an ultrasonic cell disruptor at a power of 150~300kw for 40min, with a working time of 3s and a stopping time of 3s.

9. The synthesis method according to claim 7, characterized in that, In step (2), the structural formula of the (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6 (1H,3H,5H)-trione is shown in formula (1) below: Equation (1); The preparation is carried out according to the following method: 1-Methylbarbituric acid and 2-ethylaminobenzaldehyde were added to a reaction vessel containing ethanol to construct a reaction system. The reaction system was stirred at 60-90℃ for 4-8 h. After the reaction was completed, the reaction solution was cooled to room temperature, a solid precipitated, filtered, and recrystallized with an ethanol-water mixed solvent to obtain (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione. The mass ratio of 1-methylbarbituric acid to 2-ethylaminobenzaldehyde is 1:(1~1.2); the volume mass ratio of ethanol to 1-methylbarbituric acid is (10~20):1, unit: mL / g.

10. The synthesis method according to claim 7, characterized in that, In step (2), the concentration of (5Z)-5-[(2-ethylaminophenyl)methylene]-1-methylpyrimidine-2,4,6(1H,3H,5H)-trione in solution A is 0.2~0.3 g / mL.

11. The synthesis method according to claim 7, characterized in that, In step (2), the concentration of D-glucose in solution B is 55-65 mM, the concentration of glutamate dehydrogenase is 55-65 mg / mL, and the concentration of reduced nicotinamide adenine dinucleotide phosphate is 35-45 mM.

12. The synthesis method according to claim 7, characterized in that, In step (3), the volume ratio of solution A, solution B and supernatant containing imine reductase mutant is (45~55):(440~460):10; The specific steps of terminating the reaction, extraction, and drying are as follows: first, add 5M NaOH to terminate the reaction, then extract twice with 3 times the volume of ethyl acetate, combine the extracted organic phases, and finally dry the combined organic phases with anhydrous MgSO4.

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